Targeting of chondroitin sulfate glycans

ABSTRACT

The present invention relates to functional binding fragments comprising the minimal binding fragments of VAR2CSA, to antibodies against such binding fragments of VAR2CSA, nucleic acids encoding such fragments of VAR2CSA as well as methods for their production. The invention further relates to conjugates and fusion proteins of VAR2CSA polypeptides including the minimal binding fragments and their use, in particular in the treatment of conditions associated with expression of chondroitin sulfate A (CSA), such as an inappropriate expression of chondroitin sulfate A (CSA).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/376,295, filed Aug. 1, 2014, which is a national stage filing under35 U.S.C. 371 of PCT/EP2013/052557, filed Feb. 8, 2013, whichInternational Application was published by the International Bureau inEnglish on Aug. 13, 2013, and application claims priority from U.S.Provisional Application No. 61/596,931, filed on Feb. 9, 2012, whichapplications are hereby incorporated in their entirety by reference inthis application.

FIELD OF THE INVENTION

The present invention relates to functional binding fragments comprisingthe minimal binding fragments of VAR2CSA, to antibodies against suchbinding fragments of VAR2CSA, nucleic acids encoding such fragments ofVAR2CSA as well as methods for their production. The invention furtherrelates to conjugates and fusion proteins of VAR2CSA polypeptidesincluding the minimal binding fragments and their use, in particular inthe treatment of conditions associated with expression of chondroitinsulfate A (CSA), such as an inappropriate expression of chondroitinsulfate A (CSA).

BACKGROUND OF THE INVENTION

Proteoglycans are proteins conjugated to one or more glycosaminoglycan(GAG) chains. These proteins are distributed inside cells, on the cellmembrane and in the extracellular matrix serving a variety of functions:cartilage matrix formation; the structural organization of tissues;organizations of basement membranes; regulating the role of secretoryvesicles; binding of cytokines, chemokines, growth factors, andmorphogens; protease receptors and protease inhibitors; co-receptors,tyrosine-kinase-type growth factor receptors; as endocytic receptors;facilitate cell attachment, cell-cell interactions, and cell motility aswell as cell migration.

The malaria parasite Plasmodium falciparum utilizes host cellproteoglycans in almost all stages of its complex life cycle. Thesporozoite infects hepatocytes in the liver through surface-expressedcircumsporozoite protein interacting with highly sulfated heparansulfate proteoglycans (HSPG). Merozoite infection of the erythrocytes ismediated by EBA-175 binding to sialic acid on glycophorin A. Inaddition, a number of Plasmodium falciparum Erythrocyte Membrane Protein1 (PfEMP1) proteins, mediating host endothelial adhesion, have beendescribed as glycan-binding. One of these is VAR2CSA, which is a uniquemember of the PfEMP1 protein family. VAR2CSA binds with high affinity toan unusual, low-sulfated form of chondroitin sulfate A (CSA), attachedto proteoglycans, so called Chondroitin Sulfate Proteoglycan (CSPG), inthe intervillous spaces of the placenta. VAR2CSA is a large multidomainprotein (350 kDa) expressed on the surface of P. falciparum-infectederythrocytes (IEs), and the VAR2CSA-CSA interaction is responsible forplacenta specific sequestration in placental malaria (PM). Importantly,recombinant full-length VAR2CSA ecto-domain from FCR3 and 3D7 typeparasites has shown affinity for CSA in the low nano-molar range.

CSA belongs to the family of glycosaminoglycans (GAGs), which are linearpolymers of alternating amino sugars and hexuronic acid residues,attached to proteoglycans. There are several types of GAGs including;chondroitin sulfate (CS), dermatan sulfate (DS or CSB), heparan sulfate(HS) and heparin. While the polysaccharide backbone of these GAGs issimple, considerable diversity arises in modifications such as sulfationand uronate epimerization. This is the basis for the wide variety indomain structure and biological activities of different GAGs.

CS interacts with many important factors such as growth hormones,cytokines, chemokines, and adhesion molecules and is thought to beinvolved in structural stabilization, cytokinesis, cell proliferation,differentiation, cell migration, tissue morphogenesis, organogenesis,infection, and wound repair. CS chains are composed of alternating unitsof N-acetyl-D-galactosamine (GaINAc) and glucuronic acid residues.Glucuronic acid can be sulfated at its C2 position and GaINAc can besulfated at C4 and/or C6, giving rise to various disaccharide units.Varying modifications of the sugar backbone allows structural andfunctional heterogeneity of the CS chains. Placenta adhering P.falciparum parasites specifically associate with low sulfated CSA withsulfation only at C4 of GaINAc.

Early studies pinpointed CSA as being responsible for IE sequestrationin the placenta. The specific receptor was however not known. Uponfurther research it was found that the human placenta contained threedistinct types of chondroitin sulfate proteoglycans (CSPG), but that theIE adhered specifically to low sulfated CSPG in the intervillous spaces.What is special for this type of CSPG is that only 2-8% of thedisaccharide units are C4 sulfated. In an accompanying study, aimed toidentify the specific structural requirements for the CSA, it was foundthat parasite adhesion to CSPG is inhibited by CSA containing between30-50% C4 sulfation, with the remaining 50-70% disaccharide units beingunsulfated. The minimal inhibition of binding requirements for CSA wasshown to be a dodecasaccharide (six disaccharides) containing a minimumof 2-3 or 4-5 C4 sulfated disaccharide units. Chondroitin sulfateproteoglycan 4 (CSPG4), also known as High Molecular Weight-MelanomaAssociated Antigen (HMW-MAA) or melanoma-associated chondroitin sulfateproteoglycan (MSCP), is a cell surface proteoglycan which has been shownto be expressed by melanoma cells.

CSPG4/MSCP/HMV-MAA is a large proteoglycan characterized by having CSchains on the protein backbone. The sulfation of these CS chains seemsto be primarily on C4 of GaINAc (CSA), although the degree of sulfationis not known.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide minimalfunctional binding fragments of VAR2CSA suitable for the targetingand/or detection of chondroitin sulfate glycans. Other objects ofembodiments of the invention is to provide methods for treatingconditions associated with expression, such as inappropriate expressionof the chondroitin sulfate glycans, such as CSA, wherein VAR2CSApolypeptides or fragments thereof, either alone or as part of conjugatesor fusion proteins are used to target and/or detect tissue or cellshaving an expression, such as inappropriate expression of thechondroitin sulfate glycans.

SUMMARY OF THE INVENTION

It has been found by the present inventors that VAR2CSA retains itsability to bind with high affinity and specificity to certainchondroitin sulfate proteoglycans with minimal structural elements ofthe polypeptide sequence. More importantly, the present inventors havefound that VAR2CSA polypeptides bind with high and specific affinity tocancer cells and tissues, which binding by the present inventors issuggested to be through a specific interaction with chondroitin sulfateproteoglycans expressed on the surface of the cancer cells or in thesurrounding extracellular matrix. Accordingly, the present inventorssuggest to use this specific and high affinity binding for the targetingof cancer cells or other tissues or cells with high or otherwiseexpression, such as inappropriate expression of this particular type ofchondroitin sulfate proteoglycans.

So, in a first aspect the present invention relates to an isolatedprotein fragment of VAR2CSA, which fragment consists of a sequentialamino acid sequence of

a) ID1, and

b) DBL2Xb, and optionally

c) ID2a.

In some embodiments the isolated protein fragment of VAR2CSA accordingto the present invention comprises ID2a.

In a second aspect the present invention relates to an antibody thatspecifically binds a protein fragment of VAR2CSA, which fragmentconsists of a sequential amino acid sequence of a) ID1, and b) DBL2Xb,and optionally c) ID2a. In some embodiments, the antibody according tothe present invention does not bind full length VAR2CSA polypeptides.

In a third aspect the present invention relates to nucleic acidmolecules encoding a protein fragment of VAR2CSA, which fragment consistof a sequential amino acid sequence of a) ID1, and b) DBL2Xb, andoptionally c) ID2a. The invention further relates to a nucleic acidprobe capable of hybridizing to such nucleic acid sequence understringent conditions.

In a further aspect the present invention relates to a vector comprisingan isolated nucleic acid molecule according to the invention.

In a further aspect the present invention relates to a host cellcomprising a vector comprising an isolated nucleic acid moleculeaccording to the invention.

In a further aspect the present invention relates to a method forproducing the protein fragment according to the invention, the methodcomprising cultivating a cell as defined herein in an appropriate growthmedium under conditions allowing expression of the polynucleotideconstruct and recovering the resulting protein fragment from the culturemedium.

In a further aspect the present invention relates to a conjugate orfusion protein comprising a VAR2CSA polypeptide, and a therapeutic ordiagnostic effector moiety, such as a cytotoxic moiety, fluorescentlabel, and/or a radiolabel.

It is to be understood that for a conjugate, fusion or chimeric proteincomprising a VAR2CSA polypeptide, any VAR2CSA polypeptide as definedherein may be used. Accordingly, this aspect is not limited to the useof minimal binding fragments. This applies whenever the term VAR2CSApolypeptide is used and are accordingly equally relevant when used formedical treatment, targeting or diagnosing.

In a further aspect the present invention relates to a compositioncomprising the protein fragment as defined herein, the antibodyaccording to the invention, or a conjugate according to the invention.

In a further aspect the present invention relates to a protein fragmentas defined herein, the antibody according to the invention, a VAR2CSApolypeptide, or a conjugate according to the invention for use as amedicament or diagnostic agent.

In a further aspect the present invention relates to a protein fragmentas defined herein, the antibody according to the invention, a VAR2CSApolypeptide, or a conjugate according to the invention for use indiagnosis.

In a further aspect the present invention relates to a pharmaceuticalcomposition comprising the protein fragment as defined herein, a VAR2CSApolypeptide, or a conjugate according to the invention.

In a further aspect the present invention relates to a method fordetecting a protein fragment as defined herein, or a conjugate accordingto the invention in a biological sample, said method comprising: a)obtaining a biological sample; b) contacting said biological sample withan antibody according to the invention; and c) detecting complexes ofsaid antibody and said protein fragment or conjugate, if any; as anindication of the presence of said protein fragment or conjugate in saidsample.

Accordingly, methods are provided to measure the level of proteinfragments of VAR2CSA in a biological sample. This may be used andapplied as part of a treatment, for monitoring the progress of atreatment, or alternatively as part of a production method producingVAR2CSA polypeptides according to the present invention.

In a further aspect the present invention relates to the use of aprotein fragment as defined herein, a VAR2CSA polypeptide, or aconjugate according to the invention; for the preparation of amedicament.

In a further aspect the present invention relates to a protein fragmentas defined herein, a VAR2CSA polypeptide, or a conjugate according tothe invention for the treatment of any indications associated with acondition involving expression, such as inappropriate expression of CSA,such as in cancer, arthritis, arthrosis, multiple sclerosis, healingafter neural damage, cartilage repair, wound healing, and in psoriasis.

In a further aspect the present invention relates to a method for thetreatment of any indication associated with expression, such asinappropriate expression of CSA, such as in cancer, arthritis,arthrosis, multiple sclerosis, pathological conditions caused by neuraldamage, conditions of the cartilage and scar tissue, such as inrheumatism, cartilage repair or wound healing, or in psoriasis; themethod comprising administering a therapeutically or prophylacticallyeffective amount of a protein fragment as defined herein, a VAR2CSApolypeptide, or a conjugate according to the invention to a subject inneed thereof.

In a further aspect the present invention relates to a method forpreventing the occurrence of an indication or condition associated withexpression, such as inappropriate expression of CSA, such as in cancer,multiple sclerosis, arthritis, arthrosis, pathological conditions causedby neural damage, conditions of the cartilage and scar tissue, such asin rheumatism, cartilage repair or wound healing, or in psoriasis; themethod comprising administering a therapeutically or prophylacticallyeffective amount of a protein fragment as defined herein, a VAR2CSApolypeptide, or a conjugate according to the invention to a subject inneed thereof.

In a further aspect the present invention relates to the use of aprotein fragment as defined herein, a VAR2CSA polypeptide, or aconjugate according to the invention, as a biomarker, such as tool todetect expression, such as inappropriate expression of CSA in bodyfluids such as blood, plasma, urine, saliva, feces, cerebrospinal fluid,lymph, gastric fluid, pleural fluid, cartilage fluid, sperm, and/ortissue for the diagnosis and/or prognosis of an indication or conditionassociated with expression, such as inappropriate expression of CSA,such as a malignant disease, arthritis, arthrosis, pathologicalconditions caused by neural damage, conditions of the cartilage and scartissue, such as in rheumatism or wound healing, or a cancer disease,such as brain tumors, liver tumors and tumors in the reproductive tract.

It is to be understood that as used herein the term biomarker isintended to refer to the use of VAR2CSA polypeptides, conjugates andfusion proteins according to the present invention when introduced intoan organism to detect CSA expression as a mean for diagnosis and/orprognosis of an indication or condition associated with expression ofCSA, such as inappropriate expression of CSA.

In a further aspect the present invention relates to the use of aprotein fragment as defined herein, a VAR2CSA polypeptide, or aconjugate according to the invention for the immunization of a subject,such as in a vaccine.

In a further aspect the present invention relates to the use of aprotein fragment as defined herein, a VAR2CSA polypeptide, or aconjugate according to the invention, as a targeting moiety for theisolation of a cell expressing CD44 and/or CSPG4, and/or any otherproteoglycan, such as a proteoglycan listed in Table 1.

TABLE 1 Potential molecules targeted by a VAR2CSA polypeptide GeneProtein ID 1 Protein ID 2 name NG2 CSPG4 cspg4 Neuroglycan and CSPG5 ngcNeuroglycan-C Neuropilin-1 CS NRP-1-CS NRP1 APLP2 and APP (and whenamyloid precursor- APLP2 CSA is added the proteins like protein 2 arecalled Appicans) Snorc Snorc Tomoregulin-2 TENB2 TMEFF2 Thrombomodulin,CD141 THBD Betaglycan Transforming growth factor TGFBR3 beta receptorIII Syndecan 1 CD138 SDC1 Syndecan 2 CD362 SDC2 Syndecan 3 SDC3 Syndecan4 Amphiglycan SDC4 CSPG8 CSPG8 Cd44 Glypican1-6 (kun 1 og 5) GPC1-6Brevican CSPG7 bcan lubricin Proteoglycan 4 PRG4 Dentin matrix protein 1DMP1 Neurocan CSPG3 ncan Versican CSPG2 vcan Aggrecan CSPG1 acan BamecanCSPG6 smc3 SRPX2 Sushi repeat-containing protein SRPX2 SerglycinHematopoietic proteoglycan SRGN core protein Decorin Small leucine-richproteoglycan dcn (SLRP) family Biglycan Small leucine-rich proteoglycanbgn (SLRP) family Lumican Small leucine-rich proteoglycan lum (SLRP)family Fibromodulin Small leucine-rich proteoglycan fmod (SLRP) familyKeratocan Small leucine-rich proteoglycan kera (SLRP) family Mimecanosteoglycin ogn Testican 1-3 BM-40/SPARC/osteonectin SPOCK1 family ofextracellular calcium- binding proteins. phosphacan Receptor-typetyrosine-protein PTPRZ1 phosphatase zeta Leprecan LeucineProline-Enriched LEPRE1 Proteoglycan 1 Perlecan basementmembrane-specific HSPG2 heparan sulfate proteoglycan core protein

In a further aspect the present invention relates to a method for theisolation of cells expressing CD44, and/or CSPG4 and/or any otherproteoglycan, such as a proteoglycan listed in Table 1, such as cancerstem cells in a biological sample, said method comprising:

-   -   a) obtaining a biological sample comprising cells expressing        CD44, and/or CSPG4 and/or any other proteoglycan, such as a        proteoglycan listed in Table 1;    -   b) contacting said biological sample with a protein fragment as        defined herein, a VAR2CSA polypeptide, or a conjugate according        to the invention, optionally coupled to a solid support; and    -   c) purifying or isolating the complexes of said cells expressing        CD44, and/or CSPG4 and/or any other proteoglycan, such as a        proteoglycan listed in Table 1 and said protein fragment or        conjugate.

In a further aspect the present invention relates to a diagnostic methodfor detecting elevated CSA levels in a body fluid, such as blood,plasma, urine, spinal fluid, pleura effusions, joint fluid, bone marrow,gastric fluid, faeces, semen, sperm, prostate fluid, saliva, eye fluid,lung aspirate, and lymph, in response to malignancy or other conditionsassociated with inappropriate CSA expression, the method comprising thesteps of contacting said body fluid with a protein fragment as definedherein, a VAR2CSA polypeptide, or a conjugate according to the inventionand detecting the complexes formed with CSA in said body fluid.

In a further aspect the present invention relates to a method for thepurification of CD44, and/or CSPG4, and/or any other proteoglycan, suchas a proteoglycan listed in Table 1 in a biological sample, said methodcomprising:

-   -   a) obtaining a biological sample comprising CD44, and/or CSPG4        and/or any other proteoglycan, such as a proteoglycan listed in        Table 1;    -   b) contacting said biological sample with a protein fragment as        defined herein, a VAR2CSA polypeptide, or a conjugate according        to the invention, optionally coupled to a solid support; and    -   c) purifying or isolating the complexes of said CD44, and/or        CSPG4, and/or any other proteoglycan, such as a proteoglycan        listed in Table 1 and said protein fragment or conjugate.

In a further aspect the present invention relates to a protein fragmentas defined herein, a VAR2CSA polypeptide, or a conjugate according tothe invention, or pharmaceutical composition according to the inventionin combination with any other suitable anticancer agent.

DETAILED DISCLOSURE OF THE INVENTION

This invention is based on the fact that a part of a malaria protein,the so-called VAR2CSA, can bind to a cancer specific antigen andextra-cellular CSPG with very high specificity and very high bindingstrength.

VAR2CSA mediates parasite adhesion exclusively to low-sulfatedchondroitin sulfate A (CSA) attached to proteoglycans (CSPG) in theplacenta of pregnant women. Recombinant protein has been shown to bindwith unprecedented high affinity and specificity to CSA. This may be dueto an interaction with CSA that is not only dependent on the chargedsulfates but also on the CS backbone. The inventors of the presentinvention envision that CS present in the placenta is very similar tothe CS presented on various cancer cells including cancer stem cells.This is substantiated by the fact that VAR2CSA expressing malariaparasites adhere specifically to CSA on C32 melanoma cells and to humanbrain cancer cells.

Accordingly, the current invention relies on the high affinity andspecificity between VAR2CSA recombinant proteins and low-sulfated CSA.By tagging this protein the invention can be used in a wide range ofapplications including the tracking of metastases in vivo and todiagnose metastatic disease. By coupling VAR2CSA to an apoptotic orcytotoxic reagent the invention can be used to specifically target andeliminate cancer cells and cancer stem cells. By simple therapy usingVAR2CSA recombinant protein it will be possible to neutralize theactivity of CSA thereby inhibiting tumorigenesis and/or metastasis ofCSA-expressing cancer cells. CSA can be present on a number of proteinbackbones, e.g. CSPG4, CD44, biglycan, decorin, versican, aggrecan (themajor CSPG in cartilage), perlecan, syndecan, and others listed in Table1.

The present invention is envisioned to be particularly relevant tomalignant melanoma cancer including cutaneous, ocular and conjuctivalmelanoma having CSPG4 with CSA chains on the surface of the melanomacells. This GAG chain is believed to be involved in mitoses andmetastases. However, CSPG4 is not only specific to melanoma. Micro- andtissue array analyses, performed by the inventors on data from largepanels of human tissue and cell lines, suggest that CSPG4 and othertypes of CSA-containing proteoglycans may be present on a wide range ofcancer types originating from all three cellular germ layers. Thesecancer types include carcinomas (Breast carcinoma, Pancreatic carcinoma,Ovarian carcinoma, Endometrial carcinoma, Hepatocellular carcinoma, Lungcarcinoma, Colon carcinoma, Prostate carcinoma, Cervix carcinoma, Testiscarcinoma, Basal cell skin carcinoma, Clear cell renal cell carcinoma,Kreatinized head and neck squamous cell carcinoma, Skin squamous cellcarcinoma, Vulvar kreatinized squamous cell carcinoma and Vulvar basalcell carcinoma), sarcomas (Breast liposarcoma, Fibrosarcoma,Dedifferentiated chondro- and liposarcoma, Leiomyosarcoma, Liposarcoma,Myxoid liposarcoma, Uterine corpus leiomyosarcoma, Osteosarcoma, Ewingsarcoma and Rhabdomyosarcoma), hematopoietic cancers (Chronic lymphaticleukaemia (CLL), Acute lymphatic leukaemia (ALL), Acute myeloidleukaemia (AML), B-cell, T-cell and large granular lymphoma), tumours ofneuroepithelial tissue, such as Astrocytomas (PleomorphicXanthoastrocytoma, Fibrillary Astrocytomas, Anaplastic astrocytoma,Glioblastoma Multiforme), Oligodrendroglioma, Ependymoma, Choroid plexusturmor, Oligoastrocytoma, gliosarcoma, Ganglioglioma, Retinoblastoma,Neurocytoma, Neuroblastomas (Esthesioneuroblastoma andGanglioneuroblastoma), Medulloblastoma, Atypical Teratoid Rhabdoidtumors and all types of neuroendocrine cancer.

Chondroitin sulfate proteoglycans (CSPG) also constitute an importantcomponent of the extracellular matrix of the central nerve system (CNS)including the eye and of joint cartilage. Extra-cellular CSPG iscritically involved in the pathogenesis of arthritis and the lack ofregeneration after neural damage. Loss of extra-cellular CSPG iscritical for the development of arthritis and arthrosis, and high localconcentrations of extra-cellular CSPG prevents neural out growth afterneural damage.

VAR2CSA recombinant proteins may not only be used in the treatment ofindications associated with malignant growth, such as in cancers.Therapies to either increase or decrease CSPG presence in theextracellular environment may be used to treat arthritis, athrosis andto enhance neural recovery after neurite damage, including multiplesclerosis. For these strategies the inventors of the present inventionenvision that VAR2CSA may be used either as a direct inhibitor or as amolecule targeting and maintaining drugs changing CSPG metabolism to theaffected tissues.

The inventors of the present invention have identified a malaria proteinthat binds CSA in the intervillous spaces of the placenta with anaffinity below 10 nM. Smaller recombinant parts of VAR2CSA have beenproduced at high yields that bind CSA with characteristics similar tothat of the full-length and native VAR2CSA protein. The recombinantVAR2CSA protein does not bind other CS such as chondroitin sulfate C(C6S) or highly sulfated GAGs such as heparan sulfate (HS). Recombinantproteins can be produced to bind with high affinity to CSA in variousexpression systems, here among S2 cells, T.Ni cells, CHO cells and E.coli strains including BL21 and Shuffle cells (™ Lifetechnologies).

The inventors of the present invention have also identified a singlesmall (75 kDa) antigen that binds CSA with very high affinity (nM) andhigh specificity. Table 3 (See example 2) lists the CSA affinity of allthe analyzed VAR2CSA proteins using biosensor technology.

The inventors of the present invention have shown that this VAR2CSArecombinant protein binds strongly at low concentrations to a wide rangeof cancer cell lines including cutaneous Melanoma (C32, MeWo), Lungcarcinoma (A549), Breast carcinoma (HCC1395), Osterosarcoma (U20S,MNNG/HOS), Rhabdomyosarcoma (RH30) and cutaneous T-cell lymphoma (Table4 and 5). As a control molecule another VAR2CSA protein was used, whichis identical to the minimal binding VAR2CSA construct except for a 151amino acids truncation in the C-terminal part of the molecule. Thistruncation removes the CSA binding. Recombinant VAR2CSA binds all CSPG4expressing cell lines and cancer cell lines expressing other CSPGmolecules having CSA chains (e.g. T-cell lymphoma). Recombinant VAR2CSAprotein fails to interact with human red blood cells and peripheralblood mononuclear cells (PBMC) (Table 4).

The inventors of the present invention have shown herein that malariaparasites adhere to C32 melanoma cells, probably through a specificinteraction between CSPG4 and VAR2CSA. Thus, it is envisioned thatrecombinant VAR2CSA and conjugates thereof may be used as a therapeuticcompound targeting CSA on various cancer cells.

The advantages of targeting CSA on cancer cells with VAR2CSA over othercurrent therapies in development are numerous:

1) The interaction between VAR2CSA and CSA is of unprecedented highaffinity and highly specific.2) VAR2CSA is an evolutionary refined malaria protein and it is thusunlikely that therapy will break tolerance and cause autoimmunereactions in the patient.3) VAR2CSA is a stable protein that is well characterized and can behighly expressed in organisms compatible with large-scale proteinproduction.4) VAR2CSA is a polymorphic protein with a number of serovariants.Repeated therapy could be offered by different serovariants to avoidissues with neutralizing antibodies.5) VAR2CSA is naturally exposed extracellularly on the P.falciparum-infected the red blood cell and is thus by nature a stableprotein in human serum and has been shown to be highly proteaseresistant.

The present invention is centred on the interaction between VAR2CSA andCSA. This interaction is a high affinity interaction and the main use isto target CSA expressing cancer cells.

CSA may also be involved in other diseases and pathological conditionslike for example arthritis, arthrosis, multiple sclerosis and healingafter neural damage, cartilage repair, wound healing, and in psoriasis.Accordingly, VAR2CSA polypeptides or conjugates may be used in thetreatment of any such disease or condition.

In addition the interaction between VAR2CSA and CSA could be used as abiotechnological tool, for example for protein purification and cellsorting assays.

Accordingly, the inventors of the present invention envision severaluses of this invention:

1) Traceable recombinant VAR2CSA polypeptides or conjugates may be usedto track tumors and metastases in cancer patients.2) Recombinant VAR2CSA polypeptides or conjugates may be used todirectly target and neutralize CSA activity in cancer cells.3) Recombinant VAR2CSA polypeptides or conjugates, such as VAR2CSApolypeptides coupled to a cytotoxic molecule may be used to targetcancer cells with minimal adverse toxicity to CSA-negative tissue.4) Tagged recombinant VAR2CSA polypeptides or conjugates may be used asa research or clinically developmental tool studying CSA on cancercells.5) A tagged recombinant VAR2CSA polypeptides or conjugates may be usedin assays to sort CSA-positive cells in biotechnology and life sciences.This could be done by coupling recombinant VAR2CSA to resins so that itcan be used to purify CSPG4-expressing cells, such as cancer stem cells,providing a novel and efficient biotechnological tool.6) VAR2CSA polypeptides or conjugates may be used for in vitro depletionof CSPG4-expressing cells, such as cancer cells, as part of autologoustransplantations.7) VAR2CSA polypeptides or conjugates could be used in an anti-CSPG4vaccine. By immunizing animals with CSPG4-VAR2CSA complexes orconjugates, VAR2CSA might act as a carrier and enhancer for an immuneresponse towards CSPG4 with the aim of breaking tolerance to CSPG4.8) VAR2CSA polypeptides or conjugates could be used in monitoringincreased CSA levels in body fluids (i.e. urine, spinal fluid, pleuraleffusions, joints, bone marrow, and lymph) in response to malignancy.This is based on the fact that VAR2CSA polypeptides have specificity forlow sulfated CSA and could detect tumor progression as a function of anincreased proportion of un-sulfated CS (COS).9) VAR2CSA polypeptides or conjugates could be used in treatment ofarthritis and arthrosis. The VAR2CSA polypeptides could block or targetdrugs that block protease mediated degradation of aggrecan duringarthritis and arthrosis. VAR2CSA polypeptides could also be used totarget anti-inflammatory drugs to the affected tissues and to deliverinhibitors such as ADAMTS4 and -5 inhibitors. VAR2CSA polypeptides couldbe used to target drugs that stimulate the production of aggrecan bychondrocytes. Repeated i.v. injections of aggrecan coupled to VAR2CSApolypeptides could be used to induce tolerance to aggrecan.10) VAR2CSA polypeptides or conjugates could by binding to extracellularCSPG in neural tissue inactivate the CSPGs effect on neurite outgrowthfor instance by blocking the signaling through the tyrosinephosphatase-sigma receptor. VAR2CSA peptides could target drugsdegrading CSPG or inhibiting CSPG production in affected neural tissue.For examples the following drugs could be considered to be coupled toVAR2CSA: chondroitinase ABC, which cut the sugar chains of the proteincore of CSPG molecules. Xylocides, which reduce CSPG production, ordrugs that inhibit enzymes important foe CSPG production such aschondroitin synthase or chondroitin polymerizing factor. Examples forsuch drugs include: 4-flouro-glucosamine, p-nitrophenyl-beta-D-xyloxide,4-methyl-umbelliferyl-beta-D-xylopyranoside.11) VAR2CSA polypeptides or conjugates could also be used to target andmaintain cytokines such as IL1-alfa, which stimulate production ofADAMTS4, which subsequently cleave CSPG.12) CSPG4 expression on cancer cells can influence drug resistance.Tumors in many patients usually initially respond to therapy butchemoresistance develops over time and cancer progresses. CSPG4expression is associated with multidrug resistance and is mediated byits association with integrin-induced activation of PI3K pathways.Recombinant VAR2CSA polypeptide targeting CSPG4 on cancer cells canreduce or hinder chemoresistance and could thus be used in combinationtherapies with for example PLX4032, a BRAFV600E inhibitor.

DEFINITIONS

The term “VAR2CSA polypeptide” as used herein refers to theextracellular part of a specific Erythrocyte Membrane Protein 1 (PfEMP1)protein expressed by Plasmodium falciparum interacting with chondroitinsulfate proteoglycans (CSPG) and characterized by having a sequence ofSEQ ID NO:55 or SEQ ID NO:56 or fragments or variants thereof with theability to bind chondroitin sulfate A (CSA) that could be presented on aproteoglycans (CSPG).

In some embodiments, the VAR2CSA polypeptide according to the presentinvention at least comprises the protein fragment of VAR2CSA, whichfragment consist of a sequential amino acid sequence of a) ID1, and b)DBL2Xb.

In some embodiments, the VAR2CSA polypeptide according to the presentinvention at least comprises the protein fragment of VAR2CSA, whichfragment consist of a sequential amino acid sequence of a) ID1, and b)DBL2Xb, and c) ID2a.

Included within the definition of a VAR2CSA polypeptide is polypetidesdescribed in Salanti A. et al Mol. Micro 2003 July; 49(1):179-91, inKhunrae P. et al, J Mol Biol. 2010 Apr. 2; 397(3):826-34, in SrivastavaA. et al, Proc Natl Acad Sci USA. 2010 Mar. 16; 107(11):4884-9, inDahlback M. et al, J Biol Chem. 2011 May 6; 286(18):15908-17, or inSrivastava A. et al, PLoS One. 2011; 6(5):e20270.

The term “ID1” as used herein refers to a domain of VAR2CSAcharacterized by having an amino acid sequence with at least 70%sequence identity to an amino acid sequence identified by 1-152 of SEQID NO:1.

The term “DBL2Xb” as used herein refers to a domain of VAR2CSAcharacterized by having an amino acid sequence with at least 70%sequence identity with to amino acid sequence identified by 153-577 ofSEQ ID NO:1.

The term “ID2a” as used herein refers to a domain of VAR2CSAcharacterized by having an amino acid sequence of at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, or at least 62, such as the 63 consecutive aminoacids from the N-terminal of amino acids 578-640 of SEQ ID NO:1 and withat least 70% sequence identity to such a sequence of consecutive aminoacids.

In some embodiments an amino acid sequence identity referred to hereinof at least 70% of any one sequence identified by SEQ ID NO:1-75 or afragment thereof, refers to a sequence with at least 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 8, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% sequence identity to this sequence.

The terms “variant” or “variants”, as used herein, refers to a VAR2CSApolypeptide having an amino acid sequence of SEQ ID NO:55 or SEQ IDNO:56 or a fragments a VAR2CSA polypeptide comprising an amino acidsequence of SEQ ID NO:1-54, which fragments or variants retain theability to bind chondroitin sulfate A (CSA) on proteoglycans (CSPG),wherein one or more amino acids have been substituted by another aminoacid and/or wherein one or more amino acids have been deleted and/orwherein one or more amino acids have been inserted in the polypeptideand/or wherein one or more amino acids have been added to thepolypeptide. Such addition can take place either at the N-terminal endor at the C-terminal end or both. The “variant” or “variants” withinthis definition still have functional activity in terms of being able tobind chondroitin sulfate A (CSA). In some embodiment a variant has atleast 70%, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,8, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity with the sequence of SEQ ID NO:1-75, such as thesequence of SEQ ID NO:1, 3-5, 10, 11, 29, 34, 36-38, 41, 43-45, 48,53-56, 60-70, 72-75.

The phrases “functional variant”, “functional fragment”, and “functionalderivatives” as used herein refers to variants, fragments, truncatedversions, as well as derivatives of SEQ ID NO:55 or SEQ ID NO:56, suchas any one of SEQ ID NO:1, 3-5, 10, 11, 29, 34, 36-38, 41, 43-45, 48,53-56, 60-70, 72-75, which polypeptides comprises essential bindingsequence parts of SEQ ID NO:55 or SEQ ID NO:56 and at least posses theability to bind chondroitin sulfate A (CSA). It is to be understood thata VAR2CSA functional variant or functional fragment may have two orthree features selected from being a both a variant, and/or a fragmentand/or a derivative.

A functional variant or fragment of a VAR2CSA polypeptide encompassthose that exhibit at least about 25%, such as at least about 50%, suchas at least about 75%, such as at least about 90% of the bindingaffinity of wild-type VAR2CSA polypeptide that has been produced in thesame cell type, when tested in the assays as described herein.

The term “immunologic fragment” as used herein refers to fragment of anamino acid sequence that posses essentially the same functionalactivities and the same spatial orientation to be recognized by anantibody. Accordingly a specific antibody will bind both the polypeptideand immunologic fragments thereof.

The term “another amino acid” as used herein means one amino acid thatis different from that amino acid naturally present at that position.This includes but is not limited to amino acids that can be encoded by apolynucleotide. In some embodiments the different amino acid is innatural L-form and can be encoded by a polynucleotide.

The term “derivative” as used herein, is intended to designate a VAR2CSApolypeptide exhibiting substantially the same or improved biologicalactivity relative to wild-type VAR2CSA identified by SEQ ID NO:55 or SEQID NO:56, or a fragment thereof, in which one or more of the amino acidsof the parent peptide have been chemically modified, e.g. by alkylation,PEGylation, acylation, ester formation or amide formation or the like.

The term “construct” is intended to indicate a polynucleotide segmentwhich may be based on a complete or partial naturally occurringnucleotide sequence encoding the polypeptide of interest. The constructmay optionally contain other polynucleotide segments. In a similar way,the term “amino acids which can be encoded by polynucleotide constructs”covers amino acids which can be encoded by the polynucleotide constructsdefined above, i.e. amino acids such as Ala, Val, Leu, Ile, Met, Phe,Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp and Gln.

The term “vector”, as used herein, means any nucleic acid entity capableof the amplification in a host cell. Thus, the vector may be anautonomously replicating vector, i.e. a vector, which exists as anextra-chromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated. The choice of vector will often depend onthe host cell into which it is to be introduced. Vectors include, butare not limited to plasmid vectors, phage vectors, viruses or cosmidvectors. Vectors usually contain a replication origin and at least oneselectable gene, i.e., a gene which encodes a product which is readilydetectable or the presence of which is essential for cell growth.

As used herein the term “appropriate growth medium” means a mediumcontaining nutrients and other components required for the growth ofcells and the expression of the nucleic acid sequence encoding theVAR2CSA polypeptide of the invention.

In the present context, the term “treatment” is meant to includeprevention, curing and alleviating the symptoms of a disease, disorderor condition involving expression, such as inappropriate expression ofCSA, such as in cancer. Prophylactic and therapeutic administration ofVAR2CSA polypeptide, conjugate or derivative according to the inventionis thus included in the term “treatment”.

The term “subject” as used herein means any animal, in particularmammals, such as humans, and may, where appropriate, be usedinterchangeably with the term “patient”.

The term “sequence identity” as known in the art, refers to arelationship between the sequences of two or more polypeptide moleculesor two or more nucleic acid molecules, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness between nucleic acid molecules or between polypeptides, asthe case may be, as determined by the number of matches between stringsof two or more nucleotide residues or two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity”, refers to a sequence relationship that includes bothidentical matches and conservative substitution matches. If twopolypeptide sequences have, for example, (fraction ( 10/20)) identicalamino acids, and the remainder are all non-conservative substitutions,then the percent identity and similarity would both be 50%. If, in thesame example, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% ((fraction ( 15/20))). Therefore, in cases wherethere are conservative substitutions, the degree of similarity betweentwo polypeptides will be higher than the percent identity between thosetwo polypeptides.

Conservative modifications to the amino acid sequence of SEQ ID NO:1-56, 60-70, and 72-75 (and the corresponding modifications to theencoding nucleotides) will produce VAR2CSA polypeptides havingfunctional and chemical characteristics similar to those of naturallyoccurring VAR2CSA polypeptides. In contrast, substantial modificationsin the functional and/or chemical characteristics of a VAR2CSApolypeptide may be accomplished by selecting substitutions in the aminoacid sequence of SEQ ID NO: 1-56, 60-70, and 72-75 that differsignificantly in their effect on maintaining (a) the structure of themolecular backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis” (see, forexample, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67;Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alaninescanning mutagenesis).

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of a VAR2CSApolypeptide, or to increase or decrease the affinity of a VAR2CSApolypeptide described herein.

Naturally occurring residues may be divided into classes based on commonside chain properties:

-   -   1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;    -   2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   3) acidic: Asp, Glu;    -   4) basic: His, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the Plasmodiumfalciparum VAR2CSA polypeptide that are homologous with non-Plasmodiumfalciparum VAR2CSA polypeptides, or into the non-homologous regions ofthe molecule.

In making such changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indexes are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (′3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4). In making changes based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.One may also identify epitopes from primary amino acid sequences on thebasis of hydrophilicity. These regions are also referred to as “epitopiccore regions.”

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth in SEQ ID NO:1-75 using well known techniques.For identifying suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of a VAR2CSA polypeptide to such similar polypeptides. Withsuch a comparison, one can identify residues and portions of themolecules that are conserved among similar polypeptides. It will beappreciated that changes in areas of a VAR2CSA polypeptide that are notconserved relative to such similar polypeptides would be less likely toadversely affect the biological activity and/or structure of the VAR2CSApolypeptide. One skilled in the art would also know that, even inrelatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity (conservative amino acid residue substitutions). Therefore,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a VAR2CSA polypeptide thatcorrespond to amino acid residues that are important for activity orstructure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of VAR2CSA polypeptides and other polypeptides ofthe invention.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of that information, one skilled in the art maypredict the alignment of amino acid residues of a VAR2CSA polypeptidewith respect to its three dimensional structure. One skilled in the artmay choose not to make radical changes to amino acid residues predictedto be on the surface of the protein, since such residues may be involvedin important interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. The variants can thenbe screened using activity assays as described herein. Such variantscould be used to gather information about suitable variants. Forexample, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol, 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins, which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will gain dramatically in accuracy.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-9 (1996)), “profile analysis” (Bowie et al., Science,253:164-170 (1991); Gribskov et al., Meth. Enzymol., 183:146-159 (1990);Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and“evolutionary linkage” (See Home, supra, and Brenner, supra).

Identity and similarity of related polypeptides can be readilycalculated by known methods. Such methods include, but are not limitedto, those described in Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press,New York, 1991; and Carillo et al., SIAM J. Applied Math., 48:1073(1988).

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,J. Mol. Biol., 215:403-410 (1990)). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., supra). The well known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3 times the average diagonal; the “average diagonal” is the averageof the diagonal of the comparison matrix being used; the “diagonal” isthe score or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. A standard comparison matrix (see Dayhoff et al., Atlas ofProtein Sequence and Structure, vol. 5, supp.3 (1978) for the PAM 250comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA,89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also usedby the algorithm.

Preferred parameters for a polypeptide sequence comparison include thefollowing:

Algorithm: Needleman et al., J. Mol. Biol, 48:443-453 (1970); Comparisonmatrix: BLOSUM 62 from Henikoff et al., Proc. Natl. Acad. Sci. USA,89:10915-10919 (1992); Gap Penalty: 12, Gap Length Penalty: 4, Thresholdof Similarity: 0.

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisonsinclude the following: Algorithm: Needleman et al., J. Mol Biol.,48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0, GapPenalty: 50, Gap Length Penalty: 3.

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA to DNA, protein to protein,protein to DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

The inventors of the present invention has now addressed and found theanswers to the following key questions related to the molecularmechanism behind placental adhesion in PM: 1) is the describeddifferential CSA adhesion related to the VAR2CSA sequence 2) what arethe exact minimal structural requirements for VAR2CSA binding to CSA 3)what type of chemical interaction exists between VAR2CSA and CSA andfinally 4) can this information be used to design an optimal vaccineantigen?

By expressing identical FCR3 and 3d7 VAR2CSA truncations, the presentinventors showed that VAR2CSA bind CSA with similar affinity andspecificity, regardless of parasite strain origin. These two sequenceshas a sequence identity of 79.6 To. The present inventors furtherdemonstrate that the high CSA binding-affinity is retained in severalshorter fragments, and that DBL2X, including small regions from theflanking interdomains, form a compact core that contains the highaffinity CSA binding site. In silico the present inventors definedputative GAG binding sites in VAR2CSA and by deletion and substitutionthe present inventors showed that mutations in these sites have noeffect on CSPG binding. Using the theory of polyelectrolyte-proteininteractions the present inventors have shown that the VAR2CSA-CSAinteraction may not, solely, be dependent on ionic interactions.Finally, the present inventors have shown that several short VAR2CSAfragments are capable of inducing the production of adhesion-blockingantibodies and that the anti-adhesive antibodies target the proposed CSAbinding region. These data provide the first detailed insight into thebiochemical nature of the interaction between a PfEMP1 molecule and itsligand.

Preparation of VAR2CSA Polypeptides and Other Polypeptides of theInvention

The invention also relates to a method of preparing VAR2CSA polypeptidesand other polypeptides of the invention as mentioned above. The VAR2CSApolypeptides and other polypeptides of the invention described hereinmay be produced by means of recombinant nucleic acid techniques. Ingeneral, a cloned wild-type VAR2CSA nucleic acid sequence is modified toencode the desired protein. This modified sequence is then inserted intoan expression vector, which is in turn transformed or transfected intohost cells. Higher eukaryotic cells, in particular cultured mammaliancells, may be used as host cells. Procaryotic cells such as Lactococcuslactis or E. coli can also be used to express the polypeptides as longas these prokaryotes are able to produce disulfide bonds or the proteinis or may be refolded correctly. In addition, Yeast strains can also beused to express the protein, here among Saccharomyces cerevisiae and P.Pichia.

The amino acid sequence alterations may be accomplished by a variety oftechniques. Modification of the nucleic acid sequence may be bysite-specific mutagenesis. Techniques for site-specific mutagenesis arewell known in the art and are described in, for example, Zoller andSmith (DNA 3:479-488, 1984) or “Splicing by extension overlap”, Hortonet al., Gene 77, 1989, pp. 61-68. Thus, using the nucleotide and aminoacid sequences of VAR2CSA, one may introduce the alteration(s) ofchoice. Likewise, procedures for preparing a DNA construct usingpolymerase chain reaction using specific primers are well known toper-sons skilled in the art (cf. PCR Protocols, 1990, Academic Press,San Diego, Calif., USA).

The polypeptides of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, beta-alanine,desaminohistidine, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcys-teine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, nor-valine, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into polypeptides. For example, an invitro system can be employed wherein nonsense mutations are suppressedusing chemically aminoacylated suppressor tRNAs. Methods forsynthesizing amino acids and aminoacylating tRNA are known in the art.Transcription and translation of plasmids containing nonsense mutationsis carried out in a cell-free system comprising an E. coli S30 extractand commercially available enzymes and other reagents. Polypeptides arepurified by chromatography. See, for example, Robertson et al., J. Am.Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301,1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translationis carried out in Xenopus oo-cytes by microinjection of mutated mRNA andchemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol.Chem. 271:19991-8, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the polypeptide in place ofits natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

The nucleic acid construct encoding the VAR2CSA polypeptides and otherpolypeptides of the invention of the invention may suitably be ofgenomic or cDNA origin, for instance obtained by preparing a genomic orcDNA library and screening for DNA sequences coding for all or part ofthe polypeptide by hybridization using synthetic oligonucleotide probesin accordance with standard techniques (cf. Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989).

The nucleic acid construct encoding a VAR2CSA polypeptide may also beprepared synthetically by established standard methods, e.g. thephosphoamidite method described by Beaucage and Caruthers, TetrahedronLetters 22 (1981), 1859-1869, or the method described by Matthes et al.,EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method,oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser,purified, annealed, ligated and cloned in suitable vectors. The DNAsequences encoding the Plasmodium falciparum VAR2CSA polypeptides andother polypeptides of the invention may also be prepared by polymerasechain reaction using specific primers, for instance as described in U.S.Pat. No. 4,683,202, Saiki et al., Science 239 (1988), 487-491, orSambrook et al., supra.

Furthermore, the nucleic acid construct may be of mixed synthetic andgenomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

The nucleic acid construct is preferably a DNA construct. DNA sequencesfor use in producing VAR2CSA polypeptides and other polypeptidesaccording to the present invention will typically encode a pre-propolypeptide at the amino-terminus of VAR2CSA to obtain properposttranslational processing and secretion from the host cell.

The DNA sequences encoding the Plasmodium falciparum VAR2CSApolypeptides and other polypeptides according to the present inventionare usually inserted into a recombinant vector which may be any vector,which may conveniently be subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e. a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the Plasmodium falciparum VAR2CSA polypeptides and otherpolypeptides according to the present invention is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid or viral DNA, or maycontain elements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the polypeptide.

Expression vectors for use in expressing VAR2CSA polypeptides and otherpolypeptides according to the present invention will comprise a promotercapable of directing the transcription of a cloned gene or cDNA. Thepromoter may be any DNA sequence, which shows transcriptional activityin the host cell of choice and may be derived from genes encodingproteins either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of theDNA encoding the Plasmodium falciparum VAR2CSA polypeptide in mammaliancells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981),854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al.,Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell41:521-530, 1985) or the adenovirus 2 major late promoter (Kaufman andSharp, Mol. Cell. Biol, 2:1304-1319, 1982).

An example of a suitable promoter for use in insect cells is thepolyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBSLett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen.Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosisvirus basic protein promoter (EP 397 485), the baculovirus immediateearly gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S.Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

Examples of suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No.4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654)promoters.

Examples of suitable promoters for use in filamentous fungus host cellsare, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4(1985), 2093-2099) or the tpiA promoter. Examples of other usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. niger or A.awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzaealkaline protease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters. Suitablepromoters are mentioned in, e.g. EP 238 023 and EP 383 779.

The DNA sequences encoding the Plasmodium falciparum VAR2CSApolypeptides and other polypeptides according to the present inventionmay also, if necessary, be operably connected to a suitable terminator,such as the human growth hormone terminator (Palmiter et al., Science222, 1983, pp. 809-814) or the TPI1 (Alber and Kawasaki, J. Mol. Appl.Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4,1985, pp. 2093-2099) terminators. Expression vectors may also contain aset of RNA splice sites located downstream from the promoter andupstream from the insertion site for the VAR2CSA sequence itself.Preferred RNA splice sites may be obtained from adenovirus and/orimmunoglobulin genes. Also contained in the expression vectors is apolyadenylation signal located downstream of the insertion site.Particularly preferred polyadenylation signals include the early or latepolyadenylation signal from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the adenovirus 5 Elb region, the humangrowth hormone gene terminator (DeNoto et al. Nucl. Acids Res.9:3719-3730, 1981) or the polyadenylation signal from Plasmodiumfalciparum, human or bovine genes. The expression vectors may alsoinclude a noncoding viral leader sequence, such as the adenovirus 2tripartite leader, located between the promoter and the RNA splicesites; and enhancer sequences, such as the SV40 enhancer.

To direct the Plasmodium falciparum VAR2CSA polypeptides and otherpolypeptides of the present invention into the secretory pathway of thehost cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequences encoding the Plasmodium falciparum VAR2CSA polypeptides andother polypeptides according to the present invention in the correctreading frame. Secretory signal sequences are commonly positioned 5′ tothe DNA sequence encoding the peptide. The secretory signal sequence maybe that, normally associated with the protein or may be from a geneencoding another secreted protein.

For secretion from yeast cells, the secretory signal sequence may encodeany signal peptide, which ensures efficient direction of the expressedPlasmodium falciparum VAR2CSA polypeptides and other polypeptidesaccording to the present invention into the secretory pathway of thecell. The signal peptide may be naturally occurring signal peptide, or afunctional part thereof, or it may be a synthetic peptide. Suitablesignal peptides have been found to be the alpha-factor signal peptide(cf. U.S. Pat. No. 4,870,008), the signal peptide of mouse salivaryamylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), amodified carboxypeptidase signal peptide (cf. L. A. Valls et al., Cell48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670),or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M.Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

For efficient secretion in yeast, a sequence encoding a leader peptidemay also be inserted downstream of the signal sequence and upstream ofthe DNA sequence encoding the Plasmodium falciparum VAR2CSA polypeptidesand other polypeptides according to the present invention. The functionof the leader peptide is to allow the expressed peptide to be directedfrom the endoplasmic reticulum to the Golgi apparatus and further to asecretory vesicle for secretion into the culture medium (i.e.exportation of the Plasmodium falciparum VAR2CSA polypeptides and otherpolypeptides according to the present invention across the cell wall orat least through the cellular membrane into the periplasmic space of theyeast cell). The leader peptide may be the yeast alpha-factor leader(the use of which is described in e.g. U.S. Pat. No. 4,546,082, U.S.Pat. No. 4,870,008, EP 16 201, EP 123 294, EP 123 544 and EP 163 529).Alternatively, the leader peptide may be a synthetic leader peptide,which is to say a leader peptide not found in nature. Synthetic leaderpeptides may, for instance, be constructed as described in WO 89/02463or WO 92/11378.

For use in filamentous fungi, the signal peptide may conveniently bederived from a gene encoding an Aspergillus sp. amylase or glucoamylase,a gene encoding a Rhizomucor miehei lipase or protease or a Humicolalanuginosa lipase. The signal peptide is preferably derived from a geneencoding A. oryzae TAKA amylase, A. niger neutral alpha-amylase, A.niger acid-stable amylase, or A. niger glucoamylase. Suitable signalpeptides are disclosed in, e.g. EP 238 023 and EP 215 594.

For use in insect cells, the signal peptide may conveniently be derivedfrom an insect gene (cf. WO 90/05783), such as the lepidopteran Manducasexta adipokinetic hormone precursor signal peptide (cf. U.S. Pat. No.5,023,328).

The procedures used to ligate the DNA sequences coding for thePlasmodium falciparum VAR2CSA polypeptides and other polypeptidesaccording to the present invention, the promoter and optionally theterminator and/or secretory signal sequence, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y., 1989).

Methods of transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are described in e.g. Kaufman and Sharp, J. Mol.Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1(1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982),422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson,Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52(1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass., incorporated herein by reference). The person skilledin the art will easily be able to choose suitable selectable markers.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as “carrier DNA,” to the mixture that isintroduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically 1-2 days, to begin expressing the gene ofinterest. As used herein the term “appropriate growth medium” means amedium containing nutrients and other components required for the growthof cells and the expression of the Plasmodium falciparum VAR2CSApolypeptide of interest. Media generally include a carbon source, anitrogen source, essential amino acids, essential sugars, vitamins,salts, phospholipids, protein and growth factors. Drug selection is thenapplied to select for the growth of cells that are expressing theselectable marker in a stable fashion. For cells that have beentransfected with an amplifiable selectable marker the drug concentrationmay be increased to select for an increased copy number of the clonedsequences, thereby in-creasing expression levels. Clones of stablytransfected cells are then screened for expression of the Plasmodiumfalciparum VAR2CSA polypeptide of interest.

The host cell into which the DNA sequences encoding the Plasmodiumfalciparum VAR2CSA polypeptides and other polypeptides according to thepresent invention is introduced may be any cell, which is capable ofproducing the posttranslational modified polypeptides and includesyeast, fungi and higher eucaryotic cells.

Examples of mammalian cell lines for use in the present invention arethe COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. Apreferred BHK cell line is the tk-ts13 BHK cell line (Waechter andBaserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982, incorporatedherein by reference), hereinafter referred to as BHK 570 cells. The BHK570 cell line has been deposited with the American Type CultureCollection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCCaccession number CRL 10314. A tk-ts13 BHK cell line is also availablefrom the ATCC under accession number CRL 1632. In addition, a number ofother cell lines may be used within the present invention, including RatHep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCCCCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220, 1980).

Examples of suitable yeasts cells include cells of Saccharomyces spp. orSchizosaccharomyces spp., in particular strains of Saccharomycescerevisiae or Saccharomyces kluyveri. Methods for transforming yeastcells with heterologous DNA and producing heterologous poly-peptidesthere from are described, e.g. in U.S. Pat. No. 4,599,311, U.S. Pat. No.4,931,373, U.S. Pat. Nos. 4,870,008, 5,037,743, and U.S. Pat. No.4,845,075, all of which are hereby incorporated by reference.Transformed cells are selected by a phenotype determined by a selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient, e.g. leucine. A preferred vector for use inyeast is the POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNAsequences encoding the Plasmodium falciparum VAR2CSA polypeptides andother polypeptides according to the present invention may be preceded bya signal sequence and optionally a leader sequence, e.g. as describedabove. Further examples of suitable yeast cells are strains ofKluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, orPichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132,1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).

Examples of other fungal cells are cells of filamentous fungi, e.g.Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., inparticular strains of A. oryzae, A. nidulans or A. niger. The use ofAspergillus spp. for the expression of proteins is described in, e.g.,EP 272 277, EP 238 023, EP 184 438 The transformation of F. oxysporummay, for instance, be carried out as described by Malardier et al.,1989, Gene 78: 147-156. The transformation of Trichoderma spp. may beperformed for instance as described in EP 244 234.

When a filamentous fungus is used as the host cell, it may betransformed with the DNA construct of the invention, conveniently byintegrating the DNA construct in the host chromosome to obtain arecombinant host cell. This integration is generally considered to be anadvantage as the DNA sequence is more likely to be stably maintained inthe cell. Integration of the DNA constructs into the host chromosome maybe performed according to conventional methods, e.g. by homologous orheterologous recombination.

Transformation of insect cells and production of heterologouspolypeptides therein may be performed as described in U.S. Pat. No.4,745,051; U.S. Pat. No. 4,879,236; U.S. Pat. Nos. 5,155,037; 5,162,222;EP 397,485) all of which are incorporated herein by reference. Theinsect cell line used as the host may suitably be a Lepidoptera cellline, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf.U.S. Pat. No. 5,077,214). Culture conditions may suitably be asdescribed in, for instance, WO 89/01029 or WO 89/01028, or any of theaforementioned references.

The transformed or transfected host cell described above is thencultured in a suitable nutrient medium under conditions permittingexpression of the Plasmodium falciparum VAR2CSA polypeptide after whichall or part of the resulting peptide may be recovered from the culture.The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). ThePlasmodium falciparum VAR2CSA polypeptide produced by the cells may thenbe recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaqueous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulfate,purification by a variety of chromatographic procedures, e.g. ionexchange chromatography, gelfiltration chromatography, affinitychromatography, or the like, dependent on the type of polypeptide inquestion.

Transgenic animal technology may be employed to produce the VAR2CSApolypeptides and other polypeptides of the invention. It is preferred toproduce the proteins within the mammary glands of a host female mammal.Expression in the mammary gland and subsequent secretion of the proteinof interest into the milk overcomes many difficulties encountered inisolating proteins from other sources. Milk is readily collected,available in large quantities, and biochemically well characterized.Furthermore, the major milk proteins are present in milk at highconcentrations (typically from about 1 to 15 g/l).

From a commercial point of view, it is clearly preferable to use as thehost a species that has a large milk yield. While smaller animals suchas mice and rats can be used (and are preferred at the proof ofprinciple stage), it is preferred to use livestock mammals including,but not limited to, pigs, goats, sheep and cattle. Sheep areparticularly preferred due to such factors as the previous history oftransgenesis in this species, milk yield, cost and the readyavailability of equipment for collecting sheep milk (see, for example,WO 88/00239 for a comparison of factors influencing the choice of hostspecies). It is generally desirable to select a breed of host animalthat has been bred for dairy use, such as East Friesland sheep, or tointroduce dairy stock by breeding of the transgenic line at a laterdate. In any event, animals of known, good health status should be used.

To obtain expression in the mammary gland, a transcription promoter froma milk protein gene is used. Milk protein genes include those genesencoding caseins (see U.S. Pat. No. 5,304,489), beta lactoglobulin, alactalbumin, and whey acidic protein. The beta lactoglobulin (BLG)promoter is preferred. In the case of the ovine beta lactoglobulin gene,a region of at least the proximal 406 bp of 5′ flanking sequence of thegene will generally be used, although larger portions of the 5′ flankingsequence, up to about 5 kbp, are preferred, such as a ˜4.25 kbp DNAsegment encompassing the 5′ flanking promoter and non coding portion ofthe beta lactoglobulin gene (see Whitelaw et al., Biochem. J. 286: 31 39(1992)). Similar fragments of promoter DNA from other species are alsosuitable.

Other regions of the beta lactoglobulin gene may also be incorporated inconstructs, as may genomic regions of the gene to be expressed. It isgenerally accepted in the art that constructs lacking introns, forexample, express poorly in comparison with those that contain such DNAsequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836 840(1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478 482 (1991);Whitelaw et al., Transgenic Res. 1: 3 13 (1991); WO 89/01343; and WO91/02318, each of which is incorporated herein by reference). In thisregard, it is generally preferred, where possible, to use genomicsequences containing all or some of the native introns of a geneencoding the protein or polypeptide of interest, thus the furtherinclusion of at least some introns from, e.g, the beta lactoglobulingene, is preferred. One such region is a DNA segment that provides forintron splicing and RNA polyadenylation from the 3′ non coding region ofthe ovine beta lactoglobulin gene. When substituted for the natural 3′non coding sequences of a gene, this ovine beta lactoglobulin segmentcan both enhance and stabilize expression levels of the protein orpolypeptide of interest. Within other embodiments, the regionsurrounding the initiation ATG of the VAR2CSA sequence is replaced withcorresponding sequences from a milk specific protein gene. Suchreplacement provides a putative tissue specific initiation environmentto enhance expression. It is convenient to replace the entire VAR2CSApre pro and 5′ non coding sequences with those of, for example, the BLGgene, although smaller regions may be replaced.

For expression of VAR2CSA polypeptides and other polypeptides accordingto the present invention in transgenic animals, a DNA segment encodingVAR2CSA is operably linked to additional DNA segments required for itsexpression to produce expression units. Such additional segments includethe above mentioned promoter, as well as sequences that provide fortermination of transcription and polyadenylation of mRNA. The expressionunits will further include a DNA segment encoding a secretory signalsequence operably linked to the segment encoding modified VAR2CSA. Thesecretory signal sequence may be a native secretory signal sequence ormay be that of another protein, such as a milk protein (see, forexample, von Heijne, Nucl. Acids Res. 14: 4683 4690 (1986); and Meade etal., U.S. Pat. No. 4,873,316, which are incorporated herein byreference).

Construction of expression units for use in transgenic animals isconveniently carried out by inserting a VAR2CSA sequence into a plasmidor phage vector containing the additional DNA segments, although theexpression unit may be constructed by essentially any sequence ofligations. It is particularly convenient to provide a vector containinga DNA segment encoding a milk protein and to replace the coding sequencefor the milk protein with that of a VAR2CSA variant; thereby creating agene fusion that includes the expression control sequences of the milkprotein gene. In any event, cloning of the expression units in plasmidsor other vectors facilitates the amplification of the VAR2CSA sequence.Amplification is conveniently carried out in bacterial (e.g. E. coli)host cells, thus the vectors will typically include an origin ofreplication and a selectable marker functional in bacterial host cells.The expression unit is then introduced into fertilized eggs (includingearly stage embryos) of the chosen host species. Introduction ofheterologous DNA can be accomplished by one of several routes, includingmicroinjection (e.g. U.S. Pat. No. 4,873,191), retroviral infection(Jaenisch, Science 240: 1468 1474 (1988)) or site directed integrationusing embryonic stem (ES) cells (reviewed by Bradley et al.,Bio/Technology 10: 534 539 (1992)). The eggs are then implanted into theoviducts or uteri of pseudopregnant females and allowed to develop toterm. Offspring carrying the introduced DNA in their germ line can passthe DNA on to their progeny in the normal, Mendelian fashion, allowingthe development of transgenic herds. General procedures for producingtransgenic animals are known in the art (see, for example, Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory, 1986; Simons et al., Bio/Technology 6: 179 183 (1988); Wallet al., Biol. Reprod. 32: 645 651 (1985); Buhler et al., Bio/Technology8: 140 143 (1990); Ebert et al., Bio/Technology 9: 835 838 (1991);Krimpenfort et al., Bio/Technology 9: 844 847 (1991); Wall et al., J.Cell. Biochem. 49: 113 120 (1992); U.S. Pat. No. 4,873,191; U.S. Pat.No. 4,873,316; WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458).Techniques for introducing foreign DNA sequences into mammals and theirgerm cells were originally developed in the mouse (see, e.g., Gordon etal., Proc. Natl. Acad. Sci. USA 77: 7380 7384 (1980); Gordon and Ruddle,Science 214: 1244 1246 (1981); Palmiter and Brinster, Cell 41: 343 345(1985); Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438 4442(1985); and Hogan et al. (ibid.)). These techniques were subsequentlyadapted for use with larger animals, including livestock species (see,e.g., WO 88/00239, WO 90/05188, and WO 92/11757; and Simons et al.,Bio/Technology 6: 179 183 (1988)). To summarize, in the most efficientroute used to date in the generation of transgenic mice or livestock,several hundred linear molecules of the DNA of interest are injectedinto one of the pro nuclei of a fertilized egg according to establishedtechniques. Injection of DNA into the cytoplasm of a zygote can also beemployed.

Production in transgenic plants may also be employed. Expression may begeneralised or directed to a particular organ, such as a tuber (see,Hiatt, Nature 344:469 479 (1990); Edelbaum et al., J. Interferon Res.12:449 453 (1992); Sijmons et al., Bio/Technology 8:217 221 (1990); andEP 0 255 378).

VAR2CSA Purification

The VAR2CSA polypeptides and other polypeptides of the invention may berecovered from cell culture medium or milk. The VAR2CSA polypeptides andother polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989). Preferably, they may be purified by affinity chromatographyon an anti-VAR2CSA antibody column. Additional purification may beachieved by conventional chemical purification means, such as highperformance liquid chromatography. Other methods of purification,including barium citrate precipitation, are known in the art, and may beapplied to the purification of the novel VAR2CSA polypeptides and otherpolypeptides described herein (see, for example, Scopes, R., ProteinPurification, Springer-Verlag, N.Y., 1982).

For therapeutic purposes it is preferred that the VAR2CSA polypeptidesand other polypeptides of the invention are substantially pure. Thus, ina preferred embodiment of the invention the and other polypeptides ofthe invention are purified to at least about 90 to 95% homogeneity,preferably to at least about 98% homogeneity. Purity may be assessed bye.g. gel electrophoresis and amino-terminal amino acid sequencing.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates or other materials (i.e.,contaminants) with which it is naturally associated. Preferably, theisolated polypeptide is substantially free from any other contaminatingpolypeptides or other contaminants that are found in its naturalenvironment, which would interfere with its therapeutic, diagnostic,prophylactic or research use.

The term “microorganism” as used herein refers to bacteria, fungi,archaea, protists; microscopic plants and animals (such as green algaeor plankton), the planarian and amoeba. Included within this definitionare pathogenic microorganisms.

Administration and Pharmaceutical Compositions

Combination Treatments

The VAR2CSA polypeptide, derivative, or conjugate as defined in thepresent specification may be administered simultaneously or sequentiallywith one or more other cancer agent, and/or be used in a combinationtreatment with other known therapies. The factors may be supplied insingle-dosage form wherein the single-dosage form contains bothcompounds, or in the form of a kit-of-parts comprising a preparation ofa VAR2CSA polypeptide as a first unit dosage form and a preparation ofthe one or more other compound as a second unit dosage form. Whenever afirst or second or third, etc., unit dose is mentioned throughout thisspecification this does not indicate the preferred order ofadministration, but is merely done for convenience purposes.

Suitable other cancer agents or therapies that may be used incombination with a VAR2CSA polypeptide includes antibodies already onthe market or in development, including Vemurafenib (Hoffmann-La Roche),human monoclonal antibodies against MCSP, Therapeutical (Micromet Inc)anti-MCSP using BiTE antibody platform technology, and Adoptive transferof cytotoxic T cells with specificity for MCSP.

By “simultaneous” dosing of a preparation of a VAR2CSA polypeptide and apreparation of one or more other compound is meant administration of thecompounds in single-dosage form, or administration of a first agentfollowed by administration of a second agent with a time separation ofno more than 15 minutes, preferably 10, more preferred 5, more preferred2 minutes. Either factor may be administered first.

By “sequential” dosing is meant administration of a first agent followedby administration of a second agent with a time separation of more than15 minutes. Either of the two unit dosage form may be administeredfirst. Preferably, both products are injected through the sameintravenous access.

Another object of the present invention is to provide a pharmaceuticalformulation comprising a VAR2CSA polypeptide which is present in aserum/plasma concentration from 0 mg/ml to 1 mg/ml, and wherein theformulation has a pH from 2.0 to 10.0. The formulation may furthercomprise a buffer system, preservative(s), tonicity agent(s), chelatingagent(s), stabilizers and surfactants. In some embodiments of theinvention the pharmaceutical formulation is an aqueous formulation, i.e.formulation comprising water. Such formulation is typically a solutionor a suspension. In a further embodiment of the invention thepharmaceutical formulation is an aqueous solution. The term “aqueousformulation” is defined as a formulation comprising at least 50% w/wwater. Likewise, the term “aqueous solution” is defined as a solutioncomprising at least 50% w/w water, and the term “aqueous suspension” isdefined as a suspension comprising at least 50% w/w water.

In other embodiments the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In other embodiments the pharmaceutical formulation is a driedformulation (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In a further aspect the invention relates to a pharmaceuticalformulation comprising an aqueous solution of a VAR2CSA polypeptide, anda buffer, wherein the VAR2CSA polypeptide is present in a serum/plasmaconcentration from 0-1 mg/ml or above, and wherein the formulation has apH from about 2.0 to about 10.0.

In a other embodiments of the invention the pH of the formulation isselected from the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, and 10.0.

In a further embodiment of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment of the invention the formulation furthercomprises a pharmaceutically acceptable preservative. In a furtherembodiment of the invention the preservative is selected from the groupconsisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixturesthereof. In a further embodiment of the invention the preservative ispresent in a concentration from 0.1 mg/ml to 20 mg/ml. In a furtherembodiment of the invention the preservative is present in aconcentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of theinvention the preservative is present in a concentration from 5 mg/ml to10 mg/ml. In a further embodiment of the invention the preservative ispresent in a concentration from 10 mg/ml to 20 mg/ml. Each one of thesespecific preservatives constitutes an alternative embodiment of theinvention. The use of a preservative in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises an isotonic agent. In a further embodiment of the inventionthe isotonic agent is selected from the group consisting of a salt (e.g.sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such asmono-, di-, or polysaccharides, or water-soluble glucans, including forexample fructose, glucose, mannose, sorbose, xylose, maltose, lactose,sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, solublestarch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.In some embodiments the sugar additive is sucrose. Sugar alcohol isdefined as a C4-C8 hydrocarbon having at least one —OH group andincludes, for example, mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. In some embodiments the sugar alcoholadditive is mannitol. The sugars or sugar alcohols mentioned above maybe used individually or in combination. There is no fixed limit to theamount used, as long as the sugar or sugar alcohol is soluble in theliquid preparation and does not adversely effect the stabilizing effectsachieved using the methods of the invention. In some embodiments, thesugar or sugar alcohol concentration is between about 1 mg/ml and about150 mg/ml. In a further embodiment of the invention the isotonic agentis present in a concentration from 1 mg/ml to 50 mg/ml. In a furtherembodiment of the invention the isotonic agent is present in aconcentration from 1 mg/ml to 7 mg/ml. In a further embodiment of theinvention the isotonic agent is present in a concentration from 8 mg/mlto 24 mg/ml. In a further embodiment of the invention the isotonic agentis present in a concentration from 25 mg/ml to 50 mg/ml. Each one ofthese specific isotonic agents constitutes an alternative embodiment ofthe invention. The use of an isotonic agent in pharmaceuticalcompositions is well known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises a chelating agent. In a further embodiment of the inventionthe chelating agent is selected from salts of ethylenediaminetetraaceticacid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In afurther embodiment of the invention the chelating agent is present in aconcentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of theinvention the chelating agent is present in a concentration from 0.1mg/ml to 2 mg/ml. In a further embodiment of the invention the chelatingagent is present in a concentration from 2 mg/ml to 5 mg/ml. Each one ofthese specific chelating agents constitutes an alternative embodiment ofthe invention. The use of a chelating agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises a stabilizer. The use of a stabilizer in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

More particularly, compositions of the invention are stabilized liquidpharmaceutical compositions whose therapeutically active componentsinclude a polypeptide that possibly exhibits aggregate formation duringstorage in liquid pharmaceutical formulations. By “aggregate formation”is intended a physical interaction between the polypeptide moleculesthat results in formation of oligomers, which may remain soluble, orlarge visible aggregates that precipitate from the solution. By “duringstorage” is intended a liquid pharmaceutical composition or formulationonce prepared, is not immediately administered to a subject. Rather,following preparation, it is packaged for storage, either in a liquidform, in a frozen state, or in a dried form for later reconstitutioninto a liquid form or other form suitable for administration to asubject. By “dried form” is intended the liquid pharmaceuticalcomposition or formulation is dried either by freeze drying (i.e.,lyophilization; see, for example, Williams and Polli (1984) J.Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) inSpray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez,U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm.18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), orair drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser(1991) Biopharm. 4:47-53). Aggregate formation by a polypeptide duringstorage of a liquid pharmaceutical composition can adversely affectbiological activity of that polypeptide, resulting in loss oftherapeutic efficacy of the pharmaceutical composition. Furthermore,aggregate formation may cause other problems such as blockage of tubing,membranes, or pumps when the polypeptide-containing pharmaceuticalcomposition is administered using an infusion system.

The pharmaceutical compositions of the invention may further comprise anamount of an amino acid base sufficient to decrease aggregate formationby the polypeptide during storage of the composition. By “amino acidbase” is intended an amino acid or a combination of amino acids, whereany given amino acid is present either in its free base form or in itssalt form. Where a combination of amino acids is used, all of the aminoacids may be present in their free base forms, all may be present intheir salt forms, or some may be present in their free base forms whileothers are present in their salt forms. In some embodiments, amino acidsto use in preparing the compositions of the invention are those carryinga charged side chain, such as arginine, lysine, aspartic acid, andglutamic acid. Any stereoisomer (i.e., L, D, or DL isomer) of aparticular amino acid (e.g. glycine, methionine, histidine, imidazole,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine andmixtures thereof) or combinations of these stereoisomers, may be presentin the pharmaceutical compositions of the invention so long as theparticular amino acid is present either in its free base form or itssalt form. In some embodiments the L-stereoisomer is used. Compositionsof the invention may also be formulated with analogues of these aminoacids. By “amino acid analogue” is intended a derivative of thenaturally occurring amino acid that brings about the desired effect ofdecreasing aggregate formation by the polypeptide during storage of theliquid pharmaceutical compositions of the invention. Suitable arginineanalogues include, for example, aminoguanidine, ornithine andN-monoethyl L-arginine, suitable methionine analogues include ethionineand buthionine and suitable cysteine analogues include S-methyl-Lcysteine. As with the other amino acids, the amino acid analogues areincorporated into the compositions in either their free base form ortheir salt form. In a further embodiment of the invention the aminoacids or amino acid analogues are used in a concentration, which issufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention methionine (or other sulphuricamino acids or amino acid analogous) may be added to inhibit oxidationof methionine residues to methionine sulfoxide when the polypeptideacting as the therapeutic agent is a polypeptide comprising at least onemethionine residue susceptible to such oxidation. By “inhibit” isintended minimal accumulation of methionine oxidized species over time.Inhibiting methionine oxidation results in greater retention of thepolypeptide in its proper molecular form. Any stereoisomer of methionine(L, D, or DL isomer) or combinations thereof can be used. The amount tobe added should be an amount sufficient to inhibit oxidation of themethionine residues such that the amount of methionine sulfoxide isacceptable to regulatory agencies. Typically, this means that thecomposition contains no more than about 10% to about 30% methioninesulfoxide. Generally, this can be achieved by adding methionine suchthat the ratio of methionine added to methionine residues ranges fromabout 1:1 to about 1000:1, such as 10:1 to about 100:1.

In a further embodiment of the invention the formulation furthercomprises a stabilizer selected from the group of high molecular weightpolymers or low molecular compounds. In a further embodiment of theinvention the stabilizer is selected from polyethylene glycol (e.g. PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride). Each one of these specificstabilizers constitutes an alternative embodiment of the invention.

The pharmaceutical compositions may also comprise additional stabilizingagents, which further enhance stability of a therapeutically activepolypeptide therein. Stabilizing agents of particular interest to thepresent invention include, but are not limited to, methionine and EDTA,which protect the polypeptide against methionine oxidation, and anonionic surfactant, which protects the polypeptide against aggregationassociated with freeze-thawing or mechanical shearing.

In a further embodiment of the invention the formulation furthercomprises a surfactant. In a further embodiment of the invention thesurfactant is selected from a detergent, ethoxylated castor oil,polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fattyacid esters, polyoxypropylene-polyoxyethylene block polymers (eg.poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100),polyoxyethylene sorbitan fatty acid esters, polyoxyethylene andpolyethylene derivatives such as alkylated and alkoxylated derivatives(tweens, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglyceridesor ethoxylated derivatives thereof, diglycerides or polyoxyethylenederivatives thereof, alcohols, glycerol, lectins and phospholipids (eg.phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin),derivates of phospholipids (eg. dipalmitoyl phosphatidic acid) andlysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, andglycerophospholipids (eg. cephalins), glyceroglycolipids (eg.galactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides),dodecylphosphocholine, hen egg lysolecithin, fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (eg. oleic acid and caprylic acid),acylcarnitines and derivatives, N^(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N^(α)-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N^(α)-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate),sodium caprylate, cholic acid or derivatives thereof, bile acids andsalts thereof and glycine or taurine conjugates, ursodeoxycholic acid,sodium cholate, sodium deoxycholate, sodium taurocholate, sodiumglycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationicsurfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammoniumbromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecylβ-D-glucopyranoside), poloxamines (eg. Tetronic's), which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine, or the surfactantmay be selected from the group of imidazoline derivatives, or mixturesthereof. Each one of these specific surfactants constitutes analternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing a VAR2CSA polypeptide accordingto the present invention may be administered to a patient in need ofsuch treatment at several sites, for example, at topical sites, forexample, skin and mucosal sites, at sites which bypass absorption, forexample, administration in an artery, in a vein, in the heart, and atsites which involve absorption, for example, administration in the skin,under the skin, in a muscle or in the abdomen.

Topical administration may be a particular advantage in the treatment ofconditions associated with local inflammation, such as in the treatmentof inflammation associated with burn or other conditions associated withthe skin. Accordingly, in some embodiments administration is by topicaladministration.

In some particular embodiments, eye droplets may be used in conditionsassociated with the eye, such as keratitis, such as diffuse lamellarkeratitis (DLK).

Administration of pharmaceutical compositions according to the inventionmay be through several routes of administration, for example, lingual,sublingual, buccal, in the mouth, oral, in the stomach and intestine,nasal, pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, uretal, and parenteral topatients in need of such a treatment.

Compositions of the current invention may be administered in severaldosage forms, for example, as solutions, suspensions, emulsions,microemulsions, multiple emulsion, foams, salves, pastes, plasters,ointments, tablets, coated tablets, rinses, capsules, for example, hardgelatine capsules and soft gelatine capsules, suppositories, rectalcapsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops,ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginalrings, vaginal ointments, injection solution, in situ transformingsolutions, for example in situ gelling, in situ setting, in situprecipitating, in situ crystallization, infusion solution, and implants.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the VAR2CSApolypeptide, increase bioavailability, increase solubility, decreaseadverse effects, achieve chronotherapy well known to those skilled inthe art, and increase patient compliance or any combination thereof.Examples of carriers, drug delivery systems and advanced drug deliverysystems include, but are not limited to, polymers, for example celluloseand derivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylatepolymers, polylactic and polyglycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, virus like particles, bacteria likeparticles, liquid crystals and dispersions thereof, L2 phase anddispersions there of, well known to those skilled in the art of phasebehaviour in lipid-water systems, polymeric micelles, multipleemulsions, self-emulsifying, self-microemulsifying, cyclodextrins andderivatives thereof, and dendrimers.

Compositions of the current invention are useful in the formulation ofsolids, semisolids, powder and solutions for pulmonary administration ofthe VAR2CSA polypeptide, using, for example a metered dose inhaler, drypowder inhaler and a nebulizer, all being devices well known to thoseskilled in the art.

Compositions of the current invention are specifically useful in theformulation of controlled, sustained, protracting, retarded, and slowrelease drug delivery systems. More specifically, but not limited to,compositions are useful in formulation of parenteral controlled releaseand sustained release systems (both systems leading to a many-foldreduction in number of administrations), well known to those skilled inthe art. Even more preferably, are controlled release and sustainedrelease systems administered subcutaneous. Without limiting the scope ofthe invention, examples of useful controlled release system andcompositions are hydrogels, oleaginous gels, liquid crystals, polymericmicelles, microspheres, nanoparticles,

Methods to produce controlled release systems useful for compositions ofthe current invention include, but are not limited to, crystallization,condensation, co-crystallization, precipitation, co-precipitation,emulsification, dispersion, high pressure homogenisation, encapsulation,spray drying, microencapsulating, coacervation, phase separation,solvent evaporation to produce microspheres, extrusion and supercriticalfluid processes. General reference is made to Handbook of PharmaceuticalControlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) andDrug and the Pharmaceutical Sciences vol. 99: Protein Formulation andDelivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition, which may be a solution or suspension for theadministration of the VAR2CSA polypeptide in the form of a nasal orpulmonal spray. As a still further option, the pharmaceuticalcompositions containing the VAR2CSA polypeptide of the invention canalso be adapted to transdermal administration, e.g. by needle-freeinjection or from a patch, optionally an iontophoretic patch, ortransmucosal, e.g. buccal, administration.

The term “stabilized formulation” refers to a formulation with increasedphysical stability, increased chemical stability or increased physicaland chemical stability.

The term “physical stability” of the protein formulation as used hereinrefers to the tendency of the protein to form biologically inactiveand/or insoluble aggregates of the protein as a result of exposure ofthe protein to thermo-mechanical stresses and/or interaction withinterfaces and surfaces that are destabilizing, such as hydrophobicsurfaces and interfaces. Physical stability of the aqueous proteinformulations is evaluated by means of visual inspection and/or turbiditymeasurements after exposing the formulation filled in suitablecontainers (e.g. cartridges or vials) to mechanical/physical stress(e.g. agitation) at different temperatures for various time periods.Visual inspection of the formulations is performed in a sharp focusedlight with a dark background. The turbidity of the formulation ischaracterized by a visual score ranking the degree of turbidity forinstance on a scale from 0 to 3 (a formulation showing no turbiditycorresponds to a visual score 0, and a formulation showing visualturbidity in daylight corresponds to visual score 3). A formulation isclassified physical unstable with respect to protein aggregation, whenit shows visual turbidity in daylight. Alternatively, the turbidity ofthe formulation can be evaluated by simple turbidity measurements wellknown to the skilled person. Physical stability of the aqueous proteinformulations can also be evaluated by using a spectroscopic agent orprobe of the conformational status of the protein. The probe ispreferably a small molecule that preferentially binds to a non-nativeconformer of the protein. One example of a small molecular spectroscopicprobe of protein structure is Thioflavin T. Thioflavin T is afluorescent dye that has been widely used for the detection of amyloidfibrils. In the presence of fibrils, and perhaps other proteinconfigurations as well, Thioflavin T gives rise to a new excitationmaximum at about 450 nm and enhanced emission at about 482 nm when boundto a fibril protein form. Unbound Thioflavin T is essentiallynon-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in proteinstructure from native to non-native states. For instance the“hydrophobic patch” probes that bind preferentially to exposedhydrophobic patches of a protein. The hydrophobic patches are generallyburied within the tertiary structure of a protein in its native state,but become exposed as a protein begins to unfold or denature. Examplesof these small molecular, spectroscopic probes are aromatic, hydrophobicdyes, such as antrhacene, acridine, phenanthroline or the like. Otherspectroscopic probes are metal-amino acid complexes, such as cobaltmetal complexes of hydrophobic amino acids, such as phenylalanine,leucine, isoleucine, methionine, and valine, or the like.

The term “chemical stability” of the protein formulation as used hereinrefers to chemical covalent changes in the protein structure leading toformation of chemical degradation products with potential lessbiological potency and/or potential increased immunogenic propertiescompared to the native protein structure. Various chemical degradationproducts can be formed depending on the type and nature of the nativeprotein and the environment to which the protein is exposed. Eliminationof chemical degradation can most probably not be completely avoided andincreasing amounts of chemical degradation products is often seen duringstorage and use of the protein formulation as well-known by the personskilled in the art. Most proteins are prone to deamidation, a process inwhich the side chain amide group in glutaminyl or asparaginyl residuesis hydrolysed to form a free carboxylic acid. Other degradationspathways involves formation of high molecular weight transformationproducts where two or more protein molecules are covalently bound toeach other through transamidation and/or disulfide interactions leadingto formation of covalently bound dimer, oligomer and polymer degradationproducts (Stability of Protein Pharmaceuticals, Ahern. T. J. & ManningM. C., Plenum Press, New York 1992). Oxidation (of for instancemethionine residues) can be mentioned as another variant of chemicaldegradation. The chemical stability of the protein formulation can beevaluated by measuring the amount of the chemical degradation productsat various time-points after exposure to different environmentalconditions (the formation of degradation products can often beaccelerated by for instance increasing temperature). The amount of eachindividual degradation product is often determined by separation of thedegradation products depending on molecule size and/or charge usingvarious chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

Hence, as outlined above, a “stabilized formulation” refers to aformulation with increased physical stability, increased chemicalstability or increased physical and chemical stability. In general, aformulation must be stable during use and storage (in compliance withrecommended use and storage conditions) until the expiration date isreached.

In some embodiments of the invention the pharmaceutical formulationcomprising the VAR2CSA polypeptide is stable for more than 6 weeks ofusage and for more than 3 years of storage. In other embodiments of theinvention the pharmaceutical formulation comprising the VAR2CSApolypeptide is stable for more than 4 weeks of usage and for more than 3years of storage. In a further embodiment of the invention thepharmaceutical formulation comprising the VAR2CSA polypeptide is stablefor more than 4 weeks of usage and for more than two years of storage.In an even further embodiment of the invention the pharmaceuticalformulation comprising the VAR2CSA polypeptide is stable for more than 2weeks of usage and for more than two years of storage.

Indications for Use of VAR2CSA Polypeptide and Conjugates Thereof

The VAR2CSA polypeptides or conjugates thereof may be used in a widerange of indications associated with expression, such as inappropriateexpression of CSA, such as in various cancers, such as metastaticcancers including melanomas, such as C32 melanoma, sarcomas, lungcarcinomas, oligodendrocytomas, human brain tumours including gliomas,leukaemia, such as lymphoblastic leukemia and acute myeloid leukemia,and carcinoma, such as squamous cell carcinomas and breast carcinomas,renal cell carcinomas, chondrosarcomas, and pancreatic cell carcinomas.The VAR2CSA polypeptides or conjugates thereof may also be used forcancer stem cells and accordingly target the cells before developmentinto a cancer. Other conditions associated with expression, such asinappropriate expression of CSA are conditions of the cartilage and/orthe development of scar tissue.

The VAR2CSA polypeptides or conjugates thereof may be used inidentifying, tracking and targeting distant micro-metastasis in vivo.Virtually all primary tumours, including cancers of the hematopoieticsystem, have the potential of developing into metastatic disease, whichis highly associated with poor therapeutic outcome of the patients.

The VAR2CSA polypeptides or conjugates thereof may be used to targetcompounds that prevent degradation of or repair extracellular CSPG suchas growth hormones, anti-inflammatory compounds or protein inhibitors,to cartilage tissue, joints, and neural tissue.

The VAR2CSA polypeptides or conjugates thereof may be used to targetcompounds that enhance degradation or prevent production ofextracellular CSPG such as chondroitinase ABC, which cut the sugarchains of the protein core of CSPG molecules. Xylocides, which reduceCSPG production, or drugs that inhibit enzymes important for CSPGproduction such as chondroitin synthase or chondroitin polymerizingfactor (such as 4-flouro-glucosamine, p-nitrophenyl-beta-D-xyloxide,4-methyl-umbelliferyl-beta-D-xylopyranoside), to damaged neural tissue.

VAR2CSA conjugated to a nucleic acid, here among small interfering RNA(siRNA), antisense peptide nucleic acids (PNA), small hairpin RNA(shRNA) and locked nucleic acids (LNA™), can be used to remove RNAencoding CSA presenting molecules.

Conjugates of VAR2CSA Polypeptide

Therapeutic or Diagnostic Effector Moiety, Such as Cytotoxic andDetecting Moieties

In some aspects of the present invention, there are provided VAR2CSApolypeptides, fusion protein or conjugate as defined in the presentdisclosure, further comprising a therapeutic effector moiety, such as aninflammatory agent, a steroid hormone, a cytotoxic or detecting agent ormoiety, such as an organic molecule, radionuclide, or cytotoxic enzyme.

In some aspects of the present invention, the VAR2CSA polypeptide orVAR2CSA fusion protein according to the present invention comprises asequence as defined by one or more sequences selected from SEQ ID NO57-59, and 71 or a functional variant or fragment thereof.

In some embodiments the VAR2CSA polypeptide or VAR2CSA fusion proteinaccording to the present invention may comprise a protease inhibitor,such as basic pancreatic trypsin inhibitor (BPTI) in the terminal, suchas the N-terminal of the protein sequence, such as a sequence defined bySEQ ID NO:57.

In some embodiments the VAR2CSA polypeptide or VAR2CSA fusion proteinaccording to the present invention may comprise a toxin proteinsequence, such as a sequence as defined by one or more sequencesselected from SEQ ID NO 58, 59 and 71, such as a toxin protein sequencehave an optimized to be less immunogenic, such as a sequence defined bySEQ ID NO:59. In some embodiments the signal sequence KDEL of SEQ ID NO58 or 59 is present in a VAR2CSA fusion protein according to the presentinvention and in some embodiments the signal sequence KDEL of SEQ ID NO58 or 59 is absent in a VAR2CSA fusion protein according to the presentinvention. Accordingly, the signal sequence KDEL may be optional for theconstructs according to the present invention.

Non-limiting examples of cytotoxic moieties which may be fused orconjugated to VAR2CSA polypeptides according to the invention, arechemotherapeutics selected from calicheamycin, cisplatin, adriamycin,auristatin, doxorubicin, maytansinoid, taxol, ecteinascidin,geldanamycin, methotrexate and their derivatives, and combinationsthereof and the like suitable for cancer therapy. Examples of cytotoxicproteins fused to VAR2CSA polypeptides are Pseudomonas exotoxin A,diphtheria toxin, ricin toxin, pokeweed antiviral protein, saporin,gelonin and variants hereof.

Conjugates of albumin with doxorubicin for use in cancer have beendescribed (Kratz et al, Med Chem 45: 5523-33, 2002) and with metotrexatein rheumatoid arthritis (Wunder et al, Immunol 170:4793-4801, 2003).Compounds that increase reactive oxygen species, i.e. Piperlonguminehave also been described (Raj et al, Nature 475: 231-234, 2011). Also,therapeutic enzymes, agents that induce apoptosis, and the like in orderto provide for targeted cytotoxicity, i.e. killing of tumor cells, maybe used.

The VAR2CSA polypeptides described herein may mediate killing of cellsby inducing complement dependent cytotoxicity (CDC) mediated lysis,antibody dependent cellular cytotoxicity (ADCC) mediated lysis,apoptosis, homotypic adhesion, and/or phagocytosis, such as by inducingCDC mediated lysis and/or ADCC mediated lysis. The VAR2CSA polypeptidesdescribed herein may interact with components of the immune system,preferably through ADCC or CDC. However, VAR2CSA polypeptides of theinvention may also exert an effect simply by binding to tumor antigenson the cell surface, thus, e.g. blocking proliferation of the cells.

According to the invention, the term “therapeutic effector moiety” meansany molecule, which may exert a therapeutic effect. According to theinvention, a therapeutic effector molecule is preferably selectivelyguided to a cell, which expresses CSA and includes anticancer agents,radioisotopes, toxins, cytostatic or cytolytic drugs, etc. Anticanceragents comprise, for example, Anthracyclins (doxorubicin, daunorubicin,epirubicin, idarubicin, valrubicin, mitoxantrone), Platinium andnon-platinium based alkylating agents (cisplatin, carboplatin,oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil,ifosfamide, busulfan, carmustine, dacarbazine, lomustine, procarbazine),Vinca alkaloids (vincristine, vinblastine, vinorelbine, vindesine),Taxanes (taxol and decetaxel), Topoisomerase I inhibitors (camptothecin,irinotecan, topotecan), Topoisomerase II inhibitors (amsacrine,etoposide, etoposide phosphate, teniposide and other alkaloid-derivatesnaturaly occurring in the root of American Mayapple (Podophyllumpeltatum)), Non-anthracyclin cytotoxic antibiotics (dactinomycin,bleomycin, plicamycin and mitomycins), Anti-steroids (such asaminoglutethimide), Nucleoside analogues (cytarabidine, fluorouracil andmercaptopurine), Antimetabolites (methotrexate and thioguanine),dichlorodiphenyltrichloroethane analogues (like mitotane), and reactiveoxygen species (ROS)-inducing compounds (including but not limited topiperlongumine, and beta-phenylethyl isothiocyanate). Other anticanceragents are described, for example, in Goodman and Gilman, “ThePharmacological Basis of Therapeutics”, 8th Edition, 1990, McGraw-Hill,Inc., in particular Chapter 52 (Antineoplastic Agents (Paul Calabresiand Bruce A. Chabner). Toxins may be proteins such as pokeweed antiviralprotein, cholera toxin, pertussis toxin, ricin, gelonin, abrin,diphtheria exotoxin or Pseudomonas exotoxin. Toxin residues may also behigh energy-emitting radionuclides such as cobalt-60. A VAR2CSApolypeptide may be used together with cell-penetrating peptides (CPP) tofacilitate transport of the VAR2CSA polypeptide and any thereto-linkedmolecule across cell plasma membranes. Cell-penetrating peptides havefound numerous applications in medicine as drug delivery agents in thetreatment of different diseases including cancer and virus inhibitors.Examples on CPP include but are not limited to: trans-activatingtranscriptional activator (Tat) from human immunodeficiency virus; pep-1(Chariot™); R8, azo-R8; SMoC. (Okuyama M et al. Nat Methods. 2007February; 4(2):153-9M; Soane L and Fiskum GJ Neurochem. 2005 October;95(1):230-43; Loudet A et al. Org Biomol Chem. 2008 Dec. 21;6(24):4516-22).

Radionuclides

A VAR2CSA polypeptides, a fusion protein or conjugate according to theaspects described herein coupled to a polyaminopolycarboxylate chelatormay be used to provide a radiolabeled polypeptide consisting of aradiochelate of the VAR2CSA polypeptide, fusion protein or conjugatecoupled to the chelator and a radionuclide suitable for medical imaging,the radionuclide being selected from the group consisting of ⁶¹Cu, ⁶⁴Cu,⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ¹¹⁰In, ¹¹¹In, ⁴⁴Sc, ⁸⁹Zr and ⁸⁶Y, or with aradionuclide suitable for therapy, the radionuclide being selected fromthe group consisting of ²²⁵Ac, ²¹²Bi, ²¹³Bi, ⁶⁷Cu, ¹⁶⁶Ho, ¹⁷⁷Lu, ²¹²Pb,¹⁴⁹Pm, ¹⁵³Sm ²²⁷Th and ⁹⁰Y, wherein the radionuclide is complexed withthe VAR2CSA polypeptide, such as via a chelator.

Accordingly VAR2CSA polypeptides, a fusion protein or conjugateaccording to the aspects described herein may be used for radioimagingof cancer cells, including solid tumors or metastases, such as inmelanoma patients.

In embodiments thereof, the polypeptide may also be radiolabeled withnon-metal radioisotopes using so called indirect labelling. Thus, forlabelling with for example ¹⁸F, ⁷⁶Br, different iodine isotopes and²¹¹At, intermediate “linker molecules” are used for labelling. Such alinker molecule should contain two functional moieties, one providingrapid and efficient radiolabeling, and another enabling rapid andefficient coupling to the proteins, e.g. to amine groups, or preferablyto the thiol group of a unique cysteine. For example a malemide groupreacts with thiol groups to form a stable thioether bond. The “linkermolecule” may first be reacted with the radiolabel and subsequently withthe thiol or the selenothiol group of the protein.

Other alternative detecting moieties includes fluorophores orfluorochromes such as any one selected from Hydroxycoumarin,Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, PacificOrange, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates,PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein,BODIPY-FL, TRITC, X-Rhodamine, Lissamine Rhodamine B, Texas Red,Allophycocyanin (APC), and APC-Cy7 conjugates.

Such conjugates with detecting moieties include fluorophores orfluorochromes may be used for imaging of cancer cells or tumors.

Steroid Hormones or Anti-Inflammatory Agents

In some embodiments according to the invention the VAR2CSA polypeptidesare conjugated with an anti-inflammatory agent, including steroidhormones.

Cartilage and scar tissue is known to contain CSPG in high amounts.Accordingly, it may be attractive to direct anti-inflammatory agentssuch as non-steroid anti-inflammatory compounds, disease modifyinganti-rheumatic drugs (such as methotrexate, azathioprine, sulfasalazine,ciclosporine, pennicillamine, leflunomide, or gold), biologicalanti-rheumatic drugs (such as Tumor Necrosis Factor inhibitors,interleukin-1-receptor antagonists, CD20-antibody, Insulin GrowthFactor 1) and steroid hormones or alternative compounds to such tissues.

In some embodiments according to the invention the VAR2CSA polypeptidesare conjugated with an anti-inflammatory agent, such as non-steroidanti-inflammatory compounds, disease modifying anti-rheumatic drugs(such as methotrexate, azathioprine, sulfasalazine, ciclosporine,pennicillamine, leflunomide, or gold), biological anti-rheumatic drugs(such as Tumor Necrosis Factor inhibitors, interleukin-1-receptorantagonists, CD20-antibody, Insulin Growth Factor 1) and steroidhormones or alternative compounds to such tissues.

Conjugates with CSPG4

In some embodiments according to the invention the VAR2CSA polypeptidesare conjugated with CSPG4.

It is assumed that conjugates of VAR2CSA polypeptides with CSPG4 may beused as an immunization agent. For the purpose of this use, it isassumed that the VAR2CSA polypeptide may function as a chaperone thatcould facilitate a display of CSPG4 to T-cells in a conformation thatwould provide antibodies. Accordingly, it is assumed that VAR2CSApolypeptides conjugated with CSPG4 may be used in a vaccine.

As used herein the term “CSPG4” refers to the 2322 amino acid fulllength human Chondroitin sulfate proteoglycan 4 identified by Uniprot asQ6UVK1 (CSPG4 HUMAN) as well as variants, functional fragments, andorthologs thereof. CSPG4 may also be referred to as melanoma-associatedchondroitin sulfate proteoglycan (MCSP), High Molecular Weight-MelanomaAssociated Antigen (HMW-MAA) or neuron-glial antigen 2 (NG2).

Targeting of CD44 or Other Proteoglycans

For the purpose of use of conjugates of VAR2CSA polypeptides in thetreatment of cancer indications, it is assumed that the conjugatesaccording to the present invention may be used to target not only CSPG4expressing tumor cells, but also CD44 expressing cells, such as cancerstem cells, and cells expressing proteoglycans exemplified but notlimited to those of Table 1. This targeting is mediated through thebinding to CSA on the CD44 antigen. Accordingly, the conjugatesaccording to the present invention may be used to target CSPG4 negativebut CD44 positive cells. This may be used as an alternative to orsimultaneously with the targeting of CSPG4 expressing tumor cells.

Use in Isolation of Cancer Stem Cells Through Binding to CD44, and/orCSPG4, and/or Other Proteoglycans, Such as Those in Table 1.

The specific and high affinity binding of the VAR2CSA polypeptidesaccording to the present invention, such as in the form of conjugates ofVAR2CSA polypeptides, may be used to isolate stem cells, such as cancerstem cells expressing CD44 and/or CSPG4.

Use in Isolation or Detection of Circulating Tumor Cells (CTC) ThroughBinding to CSA-Containing Proteoglycans

The specific and high affinity binding of the VAR2CSA polypeptidesaccording to the present invention, such as in the form of conjugates ofVAR2CSA polypeptides, may be used to isolate or detect CTCs ofepithelial and non-epithelial origin, which express one or moreCSA-containing proteoglycans, such as those described in Table 1.

Anti-Idiotypic Antibodies

As an alternative or supplement to the use of VAR2CSA polypeptides, itis also possible to use anti-idiotypic antibodies or even mimotopes thatmimic VAR2CSA. The technologies for preparing anti-idiotypic antibodiesthat mimic an antigen epitope are known in the art and entail provisionof a first monoclonal antibody binding VAR2CSA polypeptides followed bysubsequent production of a second antibody that binds the idiotype ofsaid first antibody. Mimotopes can be isolated from libraries of randompeptides that are screened in phage display against antibodies that bindVAR2CSA polypeptides specifically.

Anti-idiotypic antibodies may also be prepared by immunization withinhibitory host or patient derived antibodies against VAR2CSA in orderto obtain and screen for polyclonal and/or monoclonal antibodies, suchas human antibodies against and inhibiting the host derived antibodies.Although VAR2CSA generally is an evolutionary refined malaria proteinunlikely to cause autoimmune reactions in the patient, such an immunereaction cannot be excluded after a period of treatment. Ananti-idiotypic antibody used in combination with or as an alternative toVAR2CSA polypeptides may then be used.

Specific Embodiments of the Invention

As described herein the present invention relates to an isolated proteinfragment of VAR2CSA, which fragment consist of a sequential amino acidsequence of

-   -   a) ID1, and    -   b) DBL2Xb, and optionally    -   c) ID2a.

In some embodiments the isolated protein fragment of VAR2CSA accordingto the present invention comprises ID2a.

In some embodiments the isolated protein fragment of VAR2CSA accordingto the present invention do not comprise ID2a.

In some embodiments the isolated protein fragment of VAR2CSA accordingto the present invention further comprises an amino acid sequence in theN- or C-terminal, or within the sequence of the protein fragment ofVAR2CSA of not more than 100 amino acids, such as not more than 90 aminoacids, such as not more than 80 amino acids, such as not more than 70amino acids, such as not more than 60 amino acids, such as not more than50 amino acids, such as not more than 40 amino acids, such as not morethan 30 amino acids, such as not more than 20 amino acids, such as notmore than 18 amino acids, such as not more than 16 amino acids, such asnot more than 14 amino acids, such as not more than 12 amino acids, suchas not more than 10 amino acids, such as not more than 8 amino acids,such as not more than 6 amino acids, such as not more than 4 aminoacids, such as not more than 2 amino acids derived from any part of aVAR2CSA polypeptide as defined herein, which is not part of ID1, DBL2Xb,or ID2a.

In some embodiments the isolated protein fragment of VAR2CSA accordingto the present invention further comprises an amino acid sequence in theN- or C-terminal, or within the sequence of the protein fragment ofVAR2CSA of not more than 100 amino acids, such as not more than 90 aminoacids, such as not more than 80 amino acids, such as not more than 70amino acids, such as not more than 60 amino acids, such as not more than50 amino acids, such as not more than 40 amino acids, such as not morethan 30 amino acids, such as not more than 20 amino acids, such as notmore than 18 amino acids, such as not more than 16 amino acids, such asnot more than 14 amino acids, such as not more than 12 amino acids, suchas not more than 10 amino acids, such as not more than 8 amino acids,such as not more than 6 amino acids, such as not more than 4 aminoacids, such as not more than 2 amino acids, which amino acid sequence isnot derived from any part of a VAR2CSA polypeptide as defined herein.

In some embodiments the protein fragment according to the presentinvention binds chondroitin sulfate A (CSA) on proteoglycans (CSPG) withan affinity as measured by a K_(D) lower than 100 nM, such as lower than80 nM, such as lower than 70 nM, such as lower than 60 nM, such as lowerthan 50 nM, such as lower than 40 nM, such as lower than 30 nM, such aslower than 26 nM, such as lower than 24 nM, such as lower than 22 nM,such as lower than 20 nM, such as lower than 18 nM, such as lower than16 nM, such as lower than 14 nM, such as lower than 12 nM, such as lowerthan 10 nM, such as lower than 9 nM, such as lower than 8 nM, such aslower than 7 nM, such as lower than 6 nM, or lower than 4 nM.

In some embodiments the protein fragment according to the presentinvention comprises an amino acid sequence having at least 70% sequenceidentity with any one amino acid sequence of 1-577 of SEQ ID NO:1, 1-592of SEQ ID NO:3, 1-579 of SEQ ID NO:4, 1-576 of SEQ ID NO:5, 1-586 of SEQID NO:10, 1-579 of SEQ ID NO:11, 1-565 of SEQ ID NO:29, 1-584 of SEQ IDNO:34, 1-569 of SEQ ID NO:36, 1-575 of SEQ ID NO:37, 1-592 of SEQ IDNO:38, 1-603 of SEQ ID NO:41, 1-588 of SEQ ID NO:43, 1-565 of SEQ IDNO:44, 1-589 of SEQ ID NO:45, 1-573 of SEQ ID NO:48, 1-583 of SEQ IDNO:53, or 1-569 of SEQ ID NO:54.

In some embodiments the protein fragment according to the presentinvention comprises an amino acid sequence having at least 70% sequenceidentity with an amino acid sequence of 578-640 of SEQ ID NO:1, 593-656of SEQ ID NO:3, 580-643 of SEQ ID NO:4, 577-640 of SEQ ID NO:5, 587-650of SEQ ID NO:10, 580-643 of SEQ ID NO:11, 566-628 of SEQ ID NO:29,585-647 of SEQ ID NO:34, 570-632 of SEQ ID NO:36, 576-639 of SEQ IDNO:37, 593-655 of SEQ ID NO:38, 604-667 of SEQ ID NO:41, 589-652 of SEQID NO:43, 566-628 of SEQ ID NO:44, 590-653 of SEQ ID NO:45, 574-637 ofSEQ ID NO:48, 584-646 of SEQ ID NO:53, or 570-632 of SEQ ID NO:54.

In some embodiments the protein fragment according to the presentinvention comprises an amino acid sequence having at least 70% sequenceidentity with an amino acid sequence of SEQ ID NO:2, 6, 8, 9, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32,33, 35, 39, 40, 42, 46, 47, 49, 50, 51, or 52.

In some embodiments the protein fragment according to the presentinvention consists of an amino acid sequence having at least 70%sequence identity with any one amino acid sequence of 1-577 of SEQ IDNO:1, 1-592 of SEQ ID NO:3, 1-579 of SEQ ID NO:4, 1-576 of SEQ ID NO:5,1-586 of SEQ ID NO:10, 1-579 of SEQ ID NO:11, 1-565 of SEQ ID NO:29,1-584 of SEQ ID NO:34, 1-569 of SEQ ID NO:36, 1-575 of SEQ ID NO:37,1-592 of SEQ ID NO:38, 1-603 of SEQ ID NO:41, 1-588 of SEQ ID NO:43,1-565 of SEQ ID NO:44, 1-589 of SEQ ID NO:45, 1-573 of SEQ ID NO:48,1-583 of SEQ ID NO:53, or 1-569 of SEQ ID NO:54.

In some embodiments the protein fragment according to the presentinvention consists of an amino acid sequence selected from the listconsisting of SEQ ID NO:1, 3-5, 10, 11, 29, 34, 36-38, 41, 43-45, 48,53, and 54.

In some embodiments the protein fragment according to the presentinvention fragment consists of an amino acid sequence having a length ofless than 700 amino acids, such as less than 690 amino acids, such asless than 680 amino acids, such as less than 670 amino acids, such asless than 660 amino acids, such as less than 650 amino acids, such asless than 640 amino acids, such as less than 630 amino acids, such asless than 620 amino acids, such as less than 610 amino acids, such asless than 600 amino acids, such as less than 590 amino acids, such asless than 580 amino acids, such as less than 570 amino acids.

In some embodiments the protein fragment according to the presentinvention is substantially pure.

In some embodiments the protein fragment according to the presentinvention has a molecular mass of less than about 100 kDa undernon-reducing conditions on an SDS-PAGE.

In some embodiments the protein fragment according to the presentinvention is a recombinant protein.

In some embodiments the protein fragment according to the presentinvention is non-glycosylated.

The invention further relates to a protein fragment as defined herein, aVAR2CSA polypeptide, or a conjugate according to the invention for thetreatment of any indications associated with a condition involvingexpression, such as inappropriate expression of CSA, such as in cancer,arthritis, multiple sclerosis and healing after neural damage, cartilagerepair, wound healing, and in psoriasis.

In some embodiments, a VAR2CSA polypeptide, conjugate or fusion proteinis or comprises a protein fragment of VAR2CSA according to the presentinvention.

Accordingly, a VAR2CSA polypeptide, conjugate or fusion proteinaccording to the present invention may comprise an amino acid sequencewith at least 70% sequence identity to an amino acid sequence identifiedby any sequence of SEQ ID NO: 1-75.

In some embodiments a VAR2CSA polypeptide according to the presentinvention consist of an amino acid sequence selected from SEQ ID NO:60-70, 72-75.

In some embodiments, the cancer is selected from Cutaneous, Ocular orConjuctival melanoma. Carcinomas (Triple negative- and metaplasticbreast carcinoma, Pancreatic carcinoma, Ovarian carcinoma, Endometrialcarcinoma, Hepatocellular carcinoma, Lung carcinoma, Colon carcinoma,Prostate carcinoma, Cervix carcinoma, Testis carcinoma, Basal cell skincarcinoma, Clear cell renal cell carcinoma, Kreatinized head and necksquamous cell carcinoma, Skin squamous cell carcinoma, Vulvarkreatinized squamous cell carcinoma and Vulvar basal cell carcinoma),sarcomas (Breast liposarcoma, Fibrosarcoma, Dedifferentiated chondro-and liposarcoma, Leiomyosarcoma, Liposarcoma, Myxoid liposarcoma,Uterine corpus leiomyosarcoma, Osteosarcoma, Ewing sarcoma andRhabdomyosarcoma), hematopoietic cancers (Chronic lymphatic leukaemia(CLL), Acute lymphatic leukaemia (ALL), Acute myeloid leukaemia (AML),B-cell, T-cell and large granular lymphoma), tumours of neuroepithelialtissue, such as Astrocytomas (Pleomorphic Xanthoastrocytoma, FibrillaryAstrocytomas, Anaplastic astrocytoma, Glioblastoma Multiforme),Oligodrendroglioma, Ependymoma, Choroid plexus turmor, Oligoastrocytoma,gliosarcoma, Ganglioglioma, Retinoblastoma, Neurocytoma, Neuroblastomas(Esthesioneuroblastoma and Ganglioneuroblastoma), Medulloblastoma andAtypical Teratoid Rhabdoid tumors, and any other CSA-expressing cancersubtype.

In some embodiments, the cancer is selected from all CSA-expressingmalignancies including carcinomas (including but not limited to Breastcarcinoma, Pancreatic carcinoma, Ovarian carcinoma, Endometrialcarcinoma, Hepatocellular carcinoma, Lung carcinoma, Colon carcinoma,Prostate carcinoma, Cervix carcinoma, Testis carcinoma, Basal cell skincarcinoma, Clear cell renal cell carcinoma, Head and neck squamous cellcarcinoma, Skin squamous cell carcinoma, Vulvar kreatinized squamouscell carcinoma and Vulvar basal cell carcinoma), sarcomas (including butnot limited to Fibrosarcoma, Dedifferentiated chondro- and liposarcoma,Leiomyosarcoma, Liposarcoma, Myxoid liposarcoma, Uterine corpusleiomyosarcoma, Osteosarcoma, Ewing sarcoma and Rhabdomyosarcoma,Synovial sarcoma, Solitary Fibrous tumor), hematopoietic cancers(including but not limited to Chronic lymphatic leukaemia (CLL), Acutelymphatic leukaemia (ALL), Acute myeloid leukaemia (AML), B-cell, T-celland large granular lymphoma), tumours of neuroepithelial tissue, suchbut not limited to Astrocytomas (Pleomorphic Xanthoastrocytoma,Fibrillary Astrocytomas, Anaplastic astrocytoma, GlioblastomaMultiforme), Oligodrendroglioma, Ependymoma, Choroid plexus turmor,Oligoastrocytoma, gliosarcoma, Ganglioglioma, Retinoblastoma,Neurocytoma, Neuroblastomas (Esthesioneuroblastoma andGanglioneuroblastoma), Medulloblastoma, Atypical Teratoid Rhabdoidtumors and all types of neuroendocrine cancer.

Sequences, including sequences of VAR2CSA polypeptides:

>fcr3 745 amino acids |640 aa; underlined sequence corresponds to the ID1 domain of FCR3, Sequence in bold corresponds to DBL2Xb domain of FCR3. Remaining sequence is ID2a (SEQ ID NO: 1)NYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGOAOTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNODECQKKLEKVFASLTNGYK CDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLD >gi|254952610|gb|ACT97135.1|VAR2CSA [Plasmodium falciparum] | 341 aa  (SEQ ID NO: 2)KCDKCKSGTSRSRKIWTWRKSSGNKEGLQEEYANTIGLSPRTQLLYLGNLRKLENVCEDVTDINFDTKEKFLAGCLIAAFHEGKNLKKRYLEKKKGDNNSKLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQQIFGKLFRKYIKKKNISTEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCSCSGDSSSGENQTNSCDDIPTIDLIPQYLRFLQEWVEHFCEQRQAKVKDVITNCNSCKESGGTCNSDCEKKCKNKCDAYKTFIEDCKGVGGTGTAGSSWVKRWYQIYMRYSKYIEDAKRNRKAGTKSCGTSSTTNVSVSTDENKCVQS- >M24 745 amino acids |656 aa  (SEQ ID NO: 3)DYIKGDPYFAEYATKLSFILNSSDANNPSGETANHNDEVCNPNESEISSVGQAQTSDPSSNKTCNTHSSIKANKKKVCKHVKLGINNNDKVLRVCVIEDTSLSGVENCCFKDLLGILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSGTSTVNKNWIWKKSSGNKEGLQKEYANTIGLPPRTHSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIAAFHEGKNLKKRYPQNKNDDNNSKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQQIFGKLFRKYIKKNISTEQDTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNITTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKDVINSCNSCKNTSSKTKLGDTCNSDCEKKCKIECEKYKKFIEECRTAVGGTAGSSWSKRWDQIYKMYSKHIEDAKRNRKAGTKNCGITTGTISGESSGANSGVTTTENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGDDKAPWTTYTTYTTYTTTEKCNKERDKSKSQQSNTSVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWISDTSKNPKGSGSTNNDYELYTYNGVKETKLPKKLNSPKLD >KMWII 745 amino acids | 643 aa  (SEQ ID NO: 4)DYIKDDPYSKEYTTKLSFILNSSDANTSSGETANHNDEACNCNESEISSVGQAQTSGPSSNKTCITHSFIKANKKKVCKDVKLGVRENDKVLRVCVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNGSCNNKNQDECQKKLEKVFVSLTNGYKCDKCKSGTSTVNKKWIWKKSSGNEKGLQKEYANTIGLPPRTQSLYLGNLPKLGNVCEDVTDINFDTKEKFLAGCLIAAFHEGKNLKISHEKKKGDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFGKYIKKNIASDENTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNSTMCNADGSVTGSGSSCDDIPTTDFIPQYLRFLQEWVEHFCKQRQEKVNAVIENCNSCKNTSGERKIGGTCNGDCKTECKNKCEAYKNFIEDCKGGDGTAGSSWVKRWDQIYKRYSKHIEDAKRNRKAGTKSCGPSSITNASVSTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDENNCGEDNAPWTTYTTYTTTEKCNKDKKKSKSQSCNTAVVVNVPSPLGNTPHEYKYACQCKIPTTEETCDDRKEYMNQWISDTSKKQKGSGSTNNDYELYTYTGVKETKLPKKLNSPKLD >1248 745 amino acids | 640 aa  (SEQ ID NO: 5)SYVKNDPYSKEYVTKLSFILNPSDANNPSGETANHNDEACNPNESEIASVGQAQTSDRLSQKACITHSFIGANKKIVCKDVKLGVREKDKDLKICVIEDDSLRGVENCCFKDLLGILQENCSDNKSGSSSNGSCNNKNQDECQKKLDEALASLHNGYKCDKCKSGTSRSKKIWTWRKFPGNGEGLQKEYANTIGLPPRTQSLYLGNLRKLENVCKGVTDINFDTKEKFLAGCLIAAFHEGKNLKISNKKKNDDNGKKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIWLAMKHGTTCSSGSGDNGDGSVTGSGSSCDDMSTIDLIPQYLRFLQEWVEHFCKQRQEKVKDVIENCKSCKNTSGERIIGGTCGSDCKTKCKGECDAYKNFIEECKRGDGTAGSPWSKRWDQIYMRYSKYIEDAKRNRKAGTKNCGTSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDENICGDDKAPWTTYTTYTTTEKCNKETDKSKSQSCNTAVVVNVPSPLGNTPHGYKYACECKIPTTEETCDDRKEYMNQWISDTSKKPKGGRSTNNDYELYTYNGVKETKLPKKSSSSKLD >gi>254952618|gb|ACT97139.1| VAR2CSA [Plasmodium falciparum] |358 aa  (SEQ ID NO: 6)KCEKCKSEQSKKNNNIWIWRKFPGNGEGLQKEYANTIGLPPRTHSLYLGNLPKLENVCKDVKDINFDTKEKFLAGCLIAAFHEGKNLKTTYPQNKNADNNSKLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIWLAMKHGAEMNSTMCNGDGSVTGSSDSGSTTCSGDNGSISCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSCKNTSGERIIGGTCGSDCEKKCKGECDAYKKFIEECKGGGGGTGTAGSPWSKRWDQIYKRYSKYIEDAKRNRKAGTKSCGPSSTTNAAASTTESKCVQS >gi|254952592|gb|ACT97126.1| VAR2CSA [Plasmodium falciparum]| 333 aa  (SEQ ID NO: 7)KCDKCKSEQSKKNNKNWIWKQFPGNGEGLQKEYANTIGLPPRTHSLYLGNLPKLENVCKGVTDINFDTKEKFLAGCLIAAFHEGKNLKTSHEKKKGDNGKKLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQQIFGKLFRKYIKKNISAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGTTCSSGSGDNGDGSVTGSGSSCDDMPTTDFIPQYLRFLQEWVEHFCKQRQEKVNAVITNCKSCKESGGTCNSDCEKKCKDECEKYKKFIEECRTAADGTAGSSWSKRWDQIYKMYSKHIEDAKRNRKAGTKNCGTSSTTNAAENKCVQS >gi|90193467|gb|ABD92329.1|erythrocyte membrane protein 1 [Plasmodium  falciparum] | 269 aa(SEQ ID NO: 8)DYIKDDPYSKEYTTKLSFILNSSDANTSSGETANHNDEACNCNESEIASVEQASISDRSSQKAYITHSSIKTNKKKVCKYVKLGINNNDKVLRVCVIEDTSLSGVENCCFKDLLGILQENCSDNKRGSSFNDSCNNNNEEACQKKLEKVLASLTNGYKCEKCKSGTSRSKKKWIWKKSSGKEGGLQKEYANTIGLPPRTQSLYLGNLPKLENVCKGVTDINFDTKEKFLAGCLIAAFHEGKNLKPSHQNKNDDNNSKLCKDLKYSFADY >gi|254952616|gb|ACT97138.1|VAR2CSA [Plasmodium falciparum] | 333 aa  (SEQ ID NO: 9)KCDKCKSGTSRSKKKVVTWRKSSGNKEGLQKEYANTIGLPPRTHSLYLGNLRKLENVCEDVTDINFDTKEKFLAGCLIAAFHEGKNLKTTYPQNKNDDNNSKLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNISTEQHTSYSSLDELRESWWNTNKKYIWLAMKHGAEMNGTTCSCSGDSSDDIPTIDLIPQYLRFLQEWVEHFCKQRQAKVNAVINSCNSCKNTSGERKLGGTCGSECKTECKNKCDAYKEFIDGTGSGGGTGTAGSSWVKRWDQIYKRYSKYIEDAKRNRKAGSKNCGTSSTTNAAESKCVQS >hb31 745 amino acids |650 aa  (SEQ ID NO: 10)SYVKNNPYSAEYVTKLSFILNSSDANTSSETPSKYYDEVCNCNESEISSVGQAQTSGPSSNKTCITHSSIKTNKKKVCKDVKLGINNNDKVLRVCVIEDTSLSGVDNCCCQDLLGILQENCSDKNQSGSSSNGSCNNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWRKSSGNEEGLQKEYANTIGLPPRTQSLYLGNLRKLENVCKGVTDINFDTKEKFLAGCLIAAFHEGKNLKTTYPQNKKKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFRKYIKKNISTEQHTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVNAVIENCNSCKECGDTCNGECKTECEKKCKIECEKYKTFIEECVTAVGGTSGSPWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGITTGTISGESSGANSGVTTTENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADNAPWTTYTTYTTYTTTKNCDIKKKTPKSQPINTSVVVNVPSPLGNTPHGYKYACQCKIPTTEESCDDRKEYMNQWIIDTSKKQKGSGSTNNDYELYTYNGVKETKLPKKSSSSKLD >hb32 745 amino acids | 643 aa  (SEQ ID NO: 11)SYVKDDPYSAEYVTKLSFILNSSDANTSSETPSKYYDEVCNCNESEISSVGQAQTSGPSSNKTCITHSSIKTNKKKVCKDVKLGINNNDKVLRVCVIEDTSLSGVDNCCCQDLLGILQENCSDKNQSGSSSNGSCNNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWRKSSGNEEGLQKEYANTIGLPPRTQSLYLGNLPKLENVCKGVTDIIYDTKEKFLSGCLIAAFHEGKNLKTSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAGMNSTTCSGDGSVTGSGSSCDDMPTIDLIPQYLRFLQEWVEHFCKQRQEKVKDVITNCNSCKECGDTCNGECKTECKTKCKGECEKYKNFIEECNGTADGGTSGSSWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGTSSTTNAAASTTENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGEDKAPWTTYTTYTTKNCDIQKKTPKPQSCDTLVVVNVPSPLGNTPHGYKYVCECKIPTTEETCDDRKEYMNQWIIDTSKKQKGSGSTNNDYELYTYNGVQIKQAAGTLKNSKLD >gi|90193475|gb|ABD92333.1|erythrocyte membrane protein 1 [Plasmodium  falciparum] | 269 aa (SEQ ID NO: 12)NYIKGDPYSAEYATKLSFILNSSDTENASEKIQKNNDEVCNCNESEIASVEQAPISDRSSQKACITHSSIKANKKKVCKHVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKSGSSSNGSCNNNNEEICQKKLEKVLASLTNGYKCDKCKSGTSTVNKNWIWKKYSGKEGGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIAAFHEGKNLKTSNKKKNDDNNSKLCKALKYSFADY >gi|254952600|gb|ACT97130.1|VAR2CSA [Plasmodium falciparum] | 344 aa  (SEQ ID NO: 13)KCDKCKSGTSTVNKKWIWKKYSGTEGGLQEEYANTIALPPRTQSLYLGNLPKLENVCKDVTDINFDTKEKFLAGCLIAAFHEGKNLKTTYLEKKKGDNGKKNDDNNSKLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQQIFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTMCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQAKVKDVITNCNSCKECGGTCNGECKTECEKKCKGECDAYKKFIEECKGKADEGTSGSSWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTSTAESKCVQS >gi|254952598|gb|ACT97129.1|VAR2CSA [Plasmodium falciparum] | 334 aa  (SEQ ID NO: 14)KCDKCKSEQSKKNNNIWIWKKSSGTEGGLQKEYANTIALPPRTQSLYLGNLRKLENVCEDVKDINFDTKEKFLAGCLIAAFHEGKNLKKRYLEKKNGDNNSKLCKALKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQHTSYSSLDELRESWWNTNKKYIWLAMKHGTTCSSGSGDNGSISCDDIPTIDLIPQYLRFLQEWVEHFCEQRQGKVNAVIENCNSCKNTSSKTKLGGTCNGECKTECKGECDAYKEFIEKCKGTAAEGTSGSSWVKRWYQIYMRYSKYIEDAKRNRKAGTKNCGTSSTTSTAESKCVQS >gi|254952596|gb|ACT97128.1|VAR2CSA [Plasmodium falciparum] | 332 aa  (SEQ ID NO: 15)KCDKCKSEQSKKNNNIWIWKKSSGTEGGLQKEYANTIALPPRTQSLYLGNLRKLENVCEDVKDINFDTKEKFLAGCLIAAFHEGKNLKKRYLEKKNGDNNSKLCKALKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGTTCSSGSGDNGSISCDDIPTIDLIPQYLRFLQEWVEHFCEQRQEKVKDVIKNCNSCKECGGTCNGECKTECKNKCKDECDAYKKFIEECEGKAAEGTSGSSWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGTSSTTSTAENKCVQS >gi|90193465|gb|ABD92328.1|erythrocyte membrane protein 1 [Plasmodium  falciparum] | 267 aa (SEQ ID NO: 16)NYIKDDPYSAEYTTKLSFILNSSDTENASEKIQKNNDEVCNPNESGIACVELAQTSGSSSNKTCNTHSFIKANKKKVCKDVKLGINKKDKDLKICVIEDDSLRGVDNCCCQDLLGILQENCSDKNQSGSSSNGSCNNKNQEACQKKLENVFASLTNGYKCEKCKSEQSKKNNKNWIWKKYSVKEEGLQKEYANTIALPPRTQSLYLGNLPKLGNVCKGVTDINFDTKEKFLAGCLIAAFHEGKNLKTTYLQNKKKLCKALKYSFADY >gi|90193477|gb|ABD92334.1|erythrocyte membrane protein 1 [Plasmodium  falciparum] | 263 aa (SEQ ID NO: 17)DYIKGDPYFAEYATKLSFILNSSDANTSSGETANHNDEACNPNESEIASVEQASISDRSSQKACNTHSSIKANKKKECKHVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNGSCDKNSEEICQKKLDEALASLHNGYKNQKCKSEQSKKNKNKWIWKKSSGNEKGLQKEYANTIGLPPRTQSLYLGNLPKLENVCEDVTDINFDTKEKFLAGCLIAAFHEGKNLKTTYPQNKNDDNGKKLCKD >gi|254952594|gb|ACT97127.1|VAR2CSA [Plasmodium falciparum] | 338 aa  (SEQ ID NO: 18)KCDKCKSEQSKKNNNIWIWKKSSGNKKGLQKEYANTIGLPPRTQSLYLGNLPKLENVCKDVTDINFDTKEKFLAGCLIAAFHEGKNLKISNEKKNDDNGKKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFRKYIKKNNTAEQHTLYSSLDELRESWWNTNKKYIWLAMKHGTTCSSGSGDNGDGSVTGSGSSCDDMSTIDLIPQYLRFLQEWVEHFCKQRQEKVNAVIENCNSCKNTSSKTKLGGTCNGECKTECEKKCKDECEKYKEFIEECKRGDGTAGSPWVKRWDQIYMRYSKYIEDAKRNRKAGTKSCGTSAAENKCVQS >gi|254952602|gb|ACT97131.1|VAR2CSA [Plasmodium falciparum] | 341 aa  (SEQ ID NO: 19)KCDKCKSEQSKKNNNIWIWKKSSGDEKGLQKEYANTIALPPRTQSLYLGNLPKLENVCKDVTDINFDTKEKFLAGCLIAAFHEGKNLKTSHQNKNADNGKKNDDNGKKLCKALKYSFADYGDLIKGTSIWDNEYTKDLELNLQQIFGKLFRKYIKRNNTAEQHTLYSSLDELRESWWNTNKKYIWLAMKHGTTCSSGSGDNGDGSVTGSGSSCDDMSTIDLIPQYLRFLQEWVEHFCKQRQEKVKDVITNCNSCKECGGTCGSDCKTKCEAYKKFIEECNGTADGGTSGSSWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSGANSGVTTTENKCVQS >gi|254952660|gb|ACT97160.1|VAR2CSA [Plasmodium falciparum] | 352 aa  (SEQ ID NO: 20)KCEKCESEQSKKNNKYWIWKKSSGNGEGLQEEYANTIALPPRTHSLCLVCLHEKEGKKTQELKNIRTNSELLKERIIAAFHEGKNLKTSPQNKNDNGKKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQHTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTMCNADGSVTGSSDSGSTTCCGDNGSISCDDMPTIDLIPQYLRFLQEWVEHFCEQRQEKVNAVITNCKSCKECGGTCNSDCEKKCKAYKEFIEKCKGGGTEGTSGSSWSKRWDQIYKRHSKHIEDAKRNRKAGTKNCGITTGTISGESSGANSGVTTTENKCVQS >gi|254952652|gb|ACT97156.1| VAR2CSA [Plasmodium falciparum] |344 aa  (SEQ ID NO: 21)KCDKCKSGTSRSRKIWTWRKFRGNGEGLQKEYANTIGLSPRTQLLYLVCLHEKGKKTQELKNISTNSELLKEWIIAAFHEGKNLKTTYPQKKNDDNGKKLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNIASDENTSYSSLDELRESWWNTNKKYIWTAMKHGAGMNGTTCCGDGSVTGSSDSGSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCEQRQEKVKDVITNCKSCKESEKKCKNKCDAYKEFIDGTGSGGGTGTAGSSWSKRWDQIYMRYSKYIEDAKRNRKAGTKNCGTSSGANSGVTTTENKCVQS >gi|254952622|gb|ACT97141.1|VAR2CSA [Plasmodium falciparum] | 350 aa  (SEQ ID NO: 22)KCEKCKSEQSKKNNKIVVTWRKFPGNGEGLQKEYANTIGLSPRTQLLYLVCLHEKGKKTQHKTISTNSELLKEWIIAAFHEGKNLKKRYLEKKKGDNNSKLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQQIFGKLFRKYIKKNIASDENTSYSSLDELRESWWNTNKKYIWTAMKHGAGMNSTMCNGDGSVTGSSDSGSTTCSGDNGSISCDDIPTIDLIPQYLRFLQEWVEHFCEQRQEKVKDVIKNCNSCKECGGTCNGECKTECKNKCKDECEKYKNFIEVCTGGDGTAGSPWSKRVVYQIYMRYSKYIEDAKRNRKAGTKSCGTSSGANSGVTTTESKCVQS >gi|254952626|gb|ACT97143.1| VAR2CSA [Plasmodium falciparum] |359 aa  (SEQ ID NO: 23)KCEKCKSEQSKKNNKNWIWRKFPGNGEGLQKEYANTIGLPPRTHSLYLVCLHEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKKRYHQNNNSGNKKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQQIFGKLFRKYIKKNISTEQDTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSSDSGSTTCSGDNGSISCDDMPTIDLIPQYLRFLQEWVEHFCEQRQEKVKDVIENCKSCKNTSGERIIGGTCNGECKTECEKKCKAACEAYKTFIEECEGKAAEGTSGSSWSKRWYQIYMRYSKYIEDAKRNRKAGTKNCGKSSGANSGVTTTENKCVQS >gi|90193469|gb|ABD92330.1|erythrocyte membrane protein 1 [Plasmodium  falciparum] | 270 aa (SEQ ID NO: 24)NYIKDDPYSKEYVTKLSFIPNSSDANNPSGETANHNDEVCNPNESEISSVEHAQTSVLLSQKAYITHSSIKANKKKVCKYVKLGVRENDKDLKICVIEDDSLRGVENCCFKDFLRILQENCSDNKRESSSNGSCNNNNEEACEKNLDEALASLTNCYKNQKCKSGTSTVNNNKWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVVCLDEKEGKTQELKNIRTNSELLKEWIIAAFHEGKNLKKRYHQNKNDDNNSKLCKALKYSFADY >gi|254952644|gb|ACT97152.1|VAR2CSA [Plasmodium falciparum] | 334 aa  (SEQ ID NO: 25)KCDKCKSEQSKKNNKYWIWKKYSVKEGGLQKEYANTIALPPRTQSLCLVVCLDEKEGKTQELKNIRTNSELLKERIIAAFHEGKNLKTYHEKKKGDDGKKLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNNTAEQHTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNGTTCSCSGDSSNDIPTIDLIPQYLRFLQEWVEHFCEQRQAKVNAVIKNCKSCKECGGTCNGECKTECKTKCKGECEKYKEFIEKCEGQAAEGTSGSSWSKRWYQIYMRYSKYIEDAKRNRKAGTKNCGTSSGANSGVTTTEN KCVQS >gi|254952642|gb|ACT97151.1|VAR2CSA [Plasmodium falciparum] | 351 aa  (SEQ ID NO: 26)KCDKCKSEQSKKNNKNWIWKKYSGTEGGLQKEYANTIALPPRTQSLYLVCLHEKEEKTQELKNISTNSELLKEWIIAAFHEGKNLKISPQNKNDNGKNLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQQIFGKLFRKYIKKNNTAEQDTLYSSLDELRESWWNTNKKYIWTAMKHGAGMNGTTCCGDGSVTGSSDSGSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCEQRQAKVKDVIKNCNSCKECGGTCNGECKTECEKKCKGECEAYKKFIEKCNGGGGEGTSGSSWSKRWDQIYMRYSKYIEDAKRNRKAGTKNCGTSSTTNAAENKCVQS >gi|254952658|gb|ACT97159.1| VAR2CSA [Plasmodium falciparum] |353 aa  (SEQ ID NO: 27)KCDKCKSGTSTVNKKWIWKKFPGKEGGLQEEYANTIALPPRTQSLCLVVCLDEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKISNKKKNDENNSKLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGTTCSSGSGDNGDGSVTGSSDSGSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQAKVKDVIENCKSCKNTSSKTKLGDTCNSDCKTKCKVACEKYKEFIEKCVSAAGGTSGSSWVKRWDQIYMRYSKYIEDAKRNRKAGTKNCGPSSTTSTAESKCVQS >gi|254952640|gb|ACT97150.1| VAR2CSA [Plasmodium falciparum] |327 aa  (SEQ ID NO: 28)KCDKCKSGTSTVNKKWIWKKYSGKEGGLQKEYANTIGLPPRTQSLCLVCLHEKEGKTQELKNISTNSELLKEWIIAAFHEGKNLKISNKKKNDDNGKKLCKDLKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNNTAEQDTLYSSLDELRESWWNTNKKYIWTAMKHGAGMNSTTCSCSGDSSNDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVNAVITNCKSCKESGGTCNSDCEKKCKIECEKYKNFIEKCVTAAGGTSGSSWSKRWDQIYKMYSKYIEDAKRNRKAGTKNCGPSSTTNAAASTDENKCVQS >dd2full 745 amino acids |628 aa  (SEQ ID NO: 29)NYIKGDPYFAEYATKLSFILNSSDTENASETPSKYYDEACNCNESEIASVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGINNNDKVLRVCVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNGSCDKNSEEICQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQKEYANTIGLPPRTQSLCLVCLHEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKTSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQHTSYSSLDELRESWWNTNKKYIWTAMKHGAGMNGTTCSCSGDSSNDMPTIDLIPQYLRFLQEWVEHFCKQRQEKVNAVIENCNSCKESGGTCNSDCKTECKNKCEAYKEFIEDCKGGGTGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDVDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTKNCDIQKKTPKSQSCDTLVVVNVPSPLGNTPHEYKYACECKIPTTEETCDDRKEYMNQWSCGSAQTVRGRSGKDDYELYTYNGVKETKPLGTLKNSKLD >gi|254952636|gb|ACT97148.1|VAR2CSA [Plasmodium falciparum] | 350 aa  (SEQ ID NO: 30)KCEKCKSEQSKKNNKNWIWRKFRGTEGGLQEEYANTIGLPPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHQNKNSGNKENLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNNTAEQHTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNGTTCNADGSVTGSSDSGSTTCSGDNGSISCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVNAVINSCNSCKNTSSKTKLGDTCNSDCKTKCKIECEKYKTFIEKCVTAAGGTSGSPWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTSTAESKCVQS >gi|254952638|gb|ACT97149.1| VAR2CSA [Plasmodium falciparum] | 330 aa (SEQ ID NO: 31)KCDKCKSEQSKKNNKNWIWRKYSGNGEGLQKEYANTIGLPPRTHSLYLVCLHEKEGKTQELKNIRTNSELLKEWIIAAFHEGKNLKTTYLENKNDENKKKLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIVVTAMKHGAEMNGTTCSSGSGDNGSISCDDIPTIDLIPQYLRFLQEWVGHFCKQRQEKVNAVITNCNSCKESGGTCNSDCEKKCKIECEKYKKFIEECRTAAGGTSGSPWSKRWDQIYKMYSKYIEDAKRNRKAGTKNCGPSSTTSTAESKCVQS >gi|254952628|gb|ACT97144.1|VAR2CSA [Plasmodium falciparum] | 334 aa  (SEQ ID NO: 32)KCDKCKSEQSKKNNKNWIWRKYSGNGEGLQKEYANTIGLPPRTHSLYLVCLHEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKKRYPQNNNSGNKKKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIWLAMKHGAEMNGTMCNADGSVTGSGSSCDDMSTIDLIPQYLRFLQEWVEHFCEQRQAKVKDVINSCKSCKESGDTCNSDCEKKCKNKCDAYKTFIEEFCTADGGTAGSPWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGTSSGANSGVTTTENKCVQS >gi|254952630|gb|ACT97145.1|VAR2CSA [Plasmodium falciparum] | 350 aa  (SEQ ID NO: 33)KCDKCKSGTSTVNKNWIWKKYSGKEEGLQKEYANTIALPPRTHSLYLVCLHEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKTSPQNNNSGNKKKLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNNTAEQHTSYSSLDELRESWWNTNKKYIWLAMKHGAEMNGTTCCGDGSVTGSSDSGSTTCSGDNGSISCDDMPTTDFIPQYLRFLQEWVEHFCKQRQEKVKHVM ESCKSCKECGDTCNGECKTECEKKCKNKCEAYKTFIEKCVSADGGTSGSSWSKRWDQIYMRYSKYIEDAKRNRKAGTKNCGTSSTTNAAASTAENKCVQS >P13 745 amino acids | 647 aa  (SEQ ID NO: 34)DYIKDDPYSAEYATKLSFILNPSDANTSSGETANHNDEVCNCNESEIASVELAPISDSSSNKTCITHSFIGANKKKECKDVKLGVREKDKDLKICVIEDDSLRGVENCCCQDLLGILQENCSDNKSGSSSNGSCDKNSEDECQKKLENVFASLKNGYKCDKCKSGTSTVNKKWIWRKYSGNGEGLQKEYANTIGLPPRTHSLYLVCLHEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKTSHQNNNSGNKKKLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNIASDENTSYSSLDELRESWWNTNKKYIWLAMKHGAEMNSTMCNGDGSVTGSSDSGSTTCSGDNGSISCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKDVITNCKSCKESGDTCNSDCEKKCKNKCEAYKKFIEERRTAAQGTAESSWVKRWDQIYMRYSKYIEDAKRNRKAGTKSCGPSSTTNAAASTAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADNAPWTTYTTYTTTKNCDIKKKTPKPQSCDTLVVVNVPSPLGNTPHEYKYACQCRTPNKQESCDDRKEYMNQWSSGSAQTVRGRSTNNDYELYTYNGVKETKPLGTLKNSKLD >gi|254952608|gb|ACT97134.1|VAR2CSA [Plasmodium falciparum] | 341 aa  (SEQ ID NO: 35)KCDKCKSGTSTVNKKWIWRKSSGNKEGLQKEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKTSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIVVTAMKHGAGMNITTCCGDGSSGENQTNSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVNAVVTNCKSCKESGGTCNGECKTKCKNKCEVYKTFIDNVGDGTAGSPWVKRWDQIYKRYSKHIEDAKRNRKAGTKNCGITTGTISGESSGATSGVTTTENKCVQS >7g8 745 amino acids |632 aa  (SEQ ID NO: 36)NYIKDDPYSKEYVTKLSFIPNSSDANTSSEKIQKNNDEVCNPNESGISSVEQAQTSGPSSNKTCITHSSIKANKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLDEALESLHNGYKNQKCKSGTSTVNKKWIWKKSSGNKEGLQKEYANTIGLPPRTQSLYLGNLPKLENVSKGVTDIIYDTKEKFLAGCLIVSFHEGKNLKTSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFRKYIKKNISAEQDTSYSSLDELRESWWNTNKKYIWIAMKHGAGMNGTTCCGDGSSGENQTNSCDDIPTIDLIPQYLRFLQEWVEHFCEQRQAKVKDVITNCKSCKNTSGERKIGGTCNGECKTKCKNKCEAYKTFIEHCKGGDGTAGSSWVKRWDQIYKRYSKHIEDAKRNRKAGTKSCGTSTAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDENNCGEDKAPWTTYTTTKNCDIQKDKSKSQSSDTLVVVNVPSPLGNTPHGYKYACQCKIPTTEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSSSTKLD >Indo 745 amino acids |639 aa  (SEQ ID NO: 37)DYIKGDPYSAEYVTKLSFIPNSSDANNPSEKIQKNNDEVCNCNESEISSVGQASISDPSSNKTCNTHSSIKANKKKVCKDVKLGVRENDKVLKICVIEHTSLRGVDNCCFKDLLGILQEPRIDKNQSGSSSNGSCDKNSEEACEKNLEKVLASLTNGYKCDKCKSGTSRSKKKWIWKKYSGKEGGLQEEYANTIGLPPRTQSLCLVVCLDEKEGKTQELKNISTNSELLKEWIIAAFPEGKNLKPSPEKKKGDNGKKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTMCNADGSVTGSGSSCDDMPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCNSCKNTSSERKIGGTCNSDCKTECKNKCEVYKKFIEDCKGGDGTAGSSWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSTVLDDNICGEDNAPWTTYTTYTTTKNCDKDKKKSKSQSCDTLVVVNVPSPLGNTPHEYKYACECRTPNKQESCDDRKEYMNQWISDNTKNPKGSGSGKDYYELYTYNGVDVKPTTVRSSSTKLD >MC 745 amino acids | 655 aa  (SEQ ID NO: 38)DYIKGDPYFAEYATKLSFILNSSDANTSSGETANHNDEACNCNESEISSVEHASISDPSSNKTCNTHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHTSLSGVENCCFKDFLRILQENCSDNKSGSSSNGSCDKNNEEACEKNLEKVFASLTNCYKCEKCKSEQSKKNNKKWTWRKSSGNKGGLQEEYANTIGLPPRTQSLCLVVCLDEKEGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKNDDNGKKNDDNNSKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIWLAMKHGAEMNGTTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQAKVKDVIENCKSCKESGNKCKTECKNKCEAYKKFIENCKGGDGTAGSSWVKRWDQIYMRYSKYIEDAKRNRKAGTKNCGPSSITNVSASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGDDKAPWTTYTTYTTYTTYTTYTTYTTYTTTKNCDKERDKSKSQSCNTAVVVNVPSPLGNTPHEYKYACECRTPSNKELCDDRKEYMNQWSSGSAQTVRDRSGKDYYELYTYNGVKETKLPKKLNSSKLD >gi|254952650|gb|ACT97155.1|VAR2CSA [Plasmodium falciparum] | 347 aa  (SEQ ID NO: 39)KCDKCKSEQSKKNNKYWIWKKSSVKEEGLQKEYANTIALPPRTHSLCLVVCLDEKGKKTQELKNISTNSELLKERIIAAFHEGKNLKTTYLEKKNADNNSKLCKALKYSFADYGDLIKGTSIWDNEYTKDLELNLQQIFGKLFRKYIKKNNTAEQHTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNGTTCCGDGSVTGSSDSGSTTCSGDNGSISCDDMPTTDFIPQYLRFLQEWVEHFCKQRQEKVKDVIENCNSCKNNLGKTEINEKCKTECKNKCEAYKNFIEKFCTADGGTSGSPWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGTSSTTSTAENKCVQS >gi|254952648|gb|ACT97154.1|VAR2CSA [Plasmodium falciparum] | 335 aa  (SEQ ID NO: 40)KCEKCKSGTSTVNKYWIWRKSSGNKEGLQKEYANTIALPPRTHSLCLVVCLDEKEGKTQELKNISTNSELLKERIIAAFHEGENLKTSHEKKKGDDGKKNADNNSKLCKALKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNIASDENTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNGTTCSCSGDSSDDMPTTDFIPQYLRFLQEWVEHFCKQRQENVNAVIENCNSCKECGGTCNSDCEKKCKTECKNKCEAYKNFIEKFCTADGGTSGYSWSKRWDQIYKRYSKYIEDAKRNRKAGTKSCGTSSTTSTAESKCVQS >ghana2 745 amino acids |667 aa  (SEQ ID NO: 41)SYVKNNPYSKEYVTKLSFILNPSDANNPSETPSKYYDEVCNCNESGIACVGQAQTSGPSSNKTCITHSFIGANKKKVCKDVKLGVREKDKDLKICVIEDTYLSGVDNCCFKDFLGMLQENCSDNKSGSSSNGSCNNKNQDECEKNLDEALASLTNGYKCEKCKSGTSTVNKYWIWRKSSGNKEGLQKEYANTIALPPRTHSLCLVVCLDEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKTSHEKKKGDDGKKNADNNSKLCKALKYSFADYGDLIKGTSIWDNDFTKDLELNLQKIFGKLFRKYIKKNIASDENTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSSDSGSTTCCGDGSVTGSGSSCDDMPTTDFIPQYLRFLQEWVEHFCKQRQENVNAVIENCNSCKECGGTCNSDCEKKCKTECKGECDAYKEFIEKCNGGAAEGTSGSSWSKRWDQIYKRYSKYIEDAKRNRKAGTKNCGTSSTTSTAESKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDENICGADNAPWTTYTTYTTYTTYTTTEKCNKETDKSKLQQCNTSVVVNVPSPLGNTPHGYKYVCECRTPNKQETCDDRKEYMNQWISDNTKNPKGSRSTN NDYELYTYNGVQIKPTTVRSNSTKLD >gi|254952634|gb|ACT97147.1|VAR2CSA [Plasmodium falciparum] | 348 aa  (SEQ ID NO: 42)KCDKCKSEQSKKNNKNWIWKKSSGNEKGLQKEYANTIGLPPRTQSLCLVVCLDEKEGKTQELKNIRTNSELLKEWIIAAFHEGKNLKTSHEKKKGDNNSKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFRKYIKKNIASDENTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCSSGSGSTTCSSGSGSTTCSSGSGDSCDDMPTIDLIPQYLRFLQEWVEHFCKQRQEKVNAVIKNCNSCKESGGTCNGECKTECKNKCEAYKTFIEEFCTADGGTSGSPWSKRWDQIYKMYSKHIEDAKRNRKAGTKNCGPSSTTNVSVSTDENKCVQS >ghana1 745 amino acids | 652 aa  (SEQ ID NO: 43)DYIKDDPYFAEYVTKLSFILNSSDANNPSGETANHNDEVCNPNESGIASVEQAQTSDPSSNKTCNTHSSIKANKKKVCKHVKLGVRENDKDLKICVIEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGNEKGLQKEYANTIGLPPRTQSLCLVVCLDEKEGKTQELKNIRTNSELLKEWIIAAFHEGKNLKKRYPQNKNDDNNSKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFRKYIKKNISTEQDTLYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCSSGSGSTTCSSGSGSTTCSSGSGDSCDDMPTTDFIPQYLRFLQEWVEHFCKQRQEKVNAVIKNCNSCKESGGTCNGECKTECKNKCEAYKTFIEEFCTADGGTSGSPWSKRWDQIYKMYSKHIEDAKRNRKAGTKNCGPSSTTNVSVSTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGEDKAPWTTYTTYTTTKKCNKETDKSKSQSCNTAVVVNVPSPLGNTPHGYKYACECKIPTTEETCDDRKEYMNQWIIDTSKKQKGSGSGKDDYELYTYNGVDVKPTTVRSNSTKLD >V1S1 745 amino acids | 628 aa  (SEQ ID NO: 44)DYIKDDPYSAQYTTKLSFILNPSDANTSSEKIQKNNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKANKKKVCKDVKLGINNNDKVLRVCVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNGSCNNNNEEACEKNLDEAPASLHNGYKNQKCKSGTSRSKKKWIWKKSSGNEKGLQEEYANTIGLPPRTQSLCLVCLHEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKTSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWIAMKHGAGMNGTTCSCSGDSSNDMPTIDLIPQYLRFLQEWVEHFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKTFIEDCNGGGTGTAGSSWVKRWDQIYKRYSKHIEDAKRNRKAGTKNCGPSSITNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDENSCGDDKAPWTTYTTYTTTKNCDIQKDKSKSQPINTSVVVNVPSPLGNTPYRYKYACECKIPTTEESCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLD >raj116_var25 745 amino acids |653 aa  (SEQ ID NO: 45)DYIKGDPYFAEYATKLSFILNPSDTENASETPSKYYDEACNPNESEIASVEQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCFKDLLGILQENCSDNKRGSSSNDSCNNNNEEACEKNLDEALASLTNGYKCDKCKSGTSTVNKKWTWRKSSGNEEGLQKEYANTIGLPPRTQSLCLVCLHEKEGKTKHKTISTNSELLKEWIIAAFHEGKNLKTSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNGTTCSSGSGDNGDSSITGSSDSGSTTCSGDNGSISCDDIPTTDFIPQYLRFLQEWVEHFCEQRQAKVKDVINSCNSCNESGGTCNGECKTKCKDECEKYKKFIEDCNGGDGTAGSSWVKRWDQIYKRYSKHIEDAKRNRKAGTKNCGPSSITNAAASTDENKCVQSDVDSFFKHLIDIGLTTPSSYLSIVLDENSCGDDKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPHEYKYACECKIPTNEETCDDRKDYMNQWISDTSKKQKGSGSGKDYYELYTYNGVQIKQAAGRSSSTKLD >gi|31323048|gb|AAP37940.1|var2csa [Plasmodium falciparum] | 490 aa  (SEQ ID NO: 46)KCDKCKSEQSKKNNNKWIWKKYSGNGEGLQKEYANTIGLPPRTQSLCLVCLHEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKKRYPQNKNDDNNSKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTN KKYIWTAMKHGAEMNGTTCSSGSGDNGDSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQAKVKDVINSCNSCKNTSGERKIGGTCNSDCEKKCKVACDAYKTFIEECRTAVGGTAGSSWVKRWDQIYKRYSKHIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDENSCGADKAPWTTYTTYTTYTTYTTYTTTEKCNKERDKSKSQQSNTSVVVNVPSPLGNTPHEYKYACECKIPTTEETCDDRKEYMNQWIIDNTKNPKGSGSTDNDYELYTYNGVQIKQAAGRSSSTKLD >gi|254952620|gb|ACT97140.1| VAR2CSA [Plasmodium falciparum] |335 aa  (SEQ ID NO: 47)KCEKCKSGTSTVNNKWIWRKSSGKEGGLQKEYANTIGLPPRTQSLYLGNLPKLENVCKGVTDIIYDTKEKFLSGCLIAAFHEGKNLKTTYLEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTN KKYIWIAMKHGAGMNGTTCSSGSGDSSNDIPTTDFIPQYLRFLQEWVENFCEQRQAKVKPVIENCNSCKESGGTCNGECKTKCKVACDAYKKFIDGTGSGGGSRPTGIAGSSWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGPSSITNVSVSTDENKCVQS >T2C6 745 amino acids I 637 aa (SEQ ID NO: 48)NYIKDDPYSKEYVTKLSFIPNSSDANTSSEKIQKNNDEVCNPNESGISSVEQAQTSDPSSNKTCITHSSIKANKKKECKDVKLGVRENDKDLKICVIEHTSLSGVDNCCFKDFLRMLQEPRIDKNQRGSSSNGSCDKNSEEACEKNLDEALASLTNGYKCDKCKSEQSKKNNNKWIWKKFPGKEGGLQEEYANTIGLPPRTQYLCLVVCLDEKEGKTQELKNIRTNSELLKEWIIAAFHEGKNLKTTYPQKKNDDNGKKLCKDLKYSFADYGDLIKGTSIWDNEYTKNVELNLQNNFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAEMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQAKVKDVITNCNSCKESGNKCKTECKNKCKDECEKYKKFIEACGTAVGGTGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADKAPWTTYTTYTTENCDIQKKTPKSQSCDTLVVVNVPSPLGNTPHGYKYACQCRTPNKQESCDDRKEYMNQWIIDNTKNPKGSGSGKDYYELCKYNGVKETKPLGTLKNSKLD >gi|254952632|gb|ACT97146.1| VAR2CSA [Plasmodium falciparum] |330 aa  (SEQ ID NO: 49)KCDKCKSEQSKKNNNKWIWRKFPGKEGGLQKEYANTIGLPPRTQSLCLVCLHEKEGKTQHKTISTNSELLKEWIIAAFHEGKNLKTTYLEKKNAENKKKLCKALKYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAGMNGTMCNADGSVTGSGSSCDDMPTTDFIPQYLRFLQEWVEHFCKQRQAKVKDVIENCKSCKESGNKCKTECKNKCDAYKTFIEECGTAVGGTAGSSWVKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTAENKCVQS >gi|90193487|gb|ABD92339.1|erythrocyte membrane protein 1 [Plasmodium  falciparum] | 269 aa(SEQ ID NO: 50)NYIKDDPYSKEYVTKLSFILNSSDAENASETPSKYYDEACNCNESGISSVEQASISDRSSQKACNTHSFIGANKKKVCKHVKLGVRENDKDLKICVIEDDSLRGVENCCFKDFLRMLQEPRIDKNQRGSSSNDSCNNNNEEACEKNLDEALASLHNGYKNQKCKSEQSKKNNNKWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVCLHEKEGKTQHKTISTNSELLKEWIIDAFHEGKNLKTTYLEKKKGDNGKKLCKALKYSFADY >gi|254952646|gb|ACT97153.1|VAR2CSA [Plasmodium falciparum] | 347 aa  (SEQ ID NO: 51)KCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIALPPRTQSLCLVVCLHEKEGKTQHKTISTNSELLKEWIIDAFHEGKNLKTTYLEKQNADNGKKNADNNSKLCKDLKYSFADYGDLIKGTSIWDNEYTKDLELNLQQIFGKLFRKYIKKNIASDENTLYSSLDELRESWWNTNKKYIWTAMKHGAEMNGTTCSSGSGDSSSGENQTNSCDDIPTIDLIPQYLRFLQEWVEHFCEQRQAKVKDVITNCKSCKESGGTCNSDCKTKCKGECEKYKKFIEKCKGGGTEGTSGSSWVKRVVYQIYMRYSKYIEDAKRNRKAGTKSCGTSSGANSGVTTTESKCVQS >gi|90193485|gb|ABD92338.1|erythrocyte membrane protein 1 [Plasmodium  falciparum] | 269 aa (SEQ ID NO: 52)DYIKDDPYSKEYTTKLSFILNSSDANTSSEKIQKNNDEVCNPNESEISSVEQAQTSRPSSNKTCITHSSIKANKKKVCKDVKLGVRENDKVLRVCVIEHTSLSGVENCCCQDLLGILQENCSDNKRGSSSNGSCDKNSEEACEKNLDEALASLTNCYKNQKCKSEQSKKNNNKWIWKKSSGNEKGLQKEYANTIGLPPRTQSLCLVCLHEKEGKTQELKNISTNSELLKEWIIAAFHEGKNLKTTYPQNKNDDNGKKLFKDLKYSFADY >MTS1 745 amino acids |646 aa  (SEQ ID NO: 53)DYIKDDPYSKEYTTKLSFILNSSDANTSSEKIQKNNDEVCNPNESEISSVEQAQTSRPSSNKTCITHSSIKANKKKVCKDVKLGVRENDKVLRVCVIEHTSLSGVENCCCQDLLGILQENCSDNKRGSSSNGSCDKNSEEACEKNLDEALASLTNCYKNQKCKSEQSKKNNNKWIWKKSSGKEGGLQKEYANTIGLPPRTQSLYLGNLPKLENVCKGVTDINFDTKEKFLAGCLIAAFHEGKNLKTTYLEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKAFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAGMNGTTCSSGSGDSSNDIPTTDFIPQYLRFLQEWVENFCEQRQAKVKDVIENCNSCKNTSGERKIGDTCNSDCEKKCKDECEKYKKFIEDCKGGDGTAGSSWVKRWDQIYKRYSKHIEDAKRNRKAGTKNCGITTGTISGESSGATSGVTTTENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGEDNAPWTTYTTYTTEKCNKETDKSKSQQSNTAVVVNVPSPLGNTPHGYKYACECKIPTTEETCDDRKEYMNQWSCGSAQTVRDRSGKDDYELCKYNGVQIKQAAGTLKNSKLD>Q8I639 (Q8I639_PLAF7) Plasmodium falciparum (isolate 3D7), 632 aa extracellular part (SEQ ID NO: 54)NYIKGDPYFAEYATKLSFILNSSDANNPSEKIQKNNDEVCNCNESGIASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQQCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLD>Q8I639 (Q8I639 PLAF7) Plasmodium falciparum (isolate 3D7), complete 2730 aaextracellular part  (SEQ ID NO: 55)MDKSSIANKIEAYLGAKSDDSKIDQSLKADPSEVQYYGSGGDGYYLRKNICKITVNHSDSGTNDPCDRIPPPYGDNDQWKCAIILSKVSEKPENVFVPPRRQRMCINNLEKLNVDKIRDKHAFLADVLLTARNEGERIVQNHPDTNSSNVCNALERSFADIADIIRGTDLWKGTNSNLEQNLKQMFAKIRENDKVLQDKYPKDQNYRKLREDWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSNGDNKLELCRKCGHYEEKVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTSEDHKSKEGTSYCSTCKDKCKKYCECVKKWKSEWENQKNKYTELYQQNKNETSQKNTSRYDDYVKDFFKKLEANYSSLENYIKGDPYFAEYATKLSFILNSSDANNPSEKIQKNNDEVCNCNESGIASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQQCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDDKDVTFFNLFEQWNKEIQYQIEQYMTNTKISCNNEKNVLSRVSDEAAQPKFSDNERDRNSITHEDKNCKEKCKCYSLWIEKINDQWDKQKDNYNKFQRKQIYDANKGSQNKKVVSLSNFLFFSCWEEYIQKYFNGDWSKIKNIGSDTFEFLIKKCGNDSGDGETIFSEKLNNAEKKCKENESTNNKMKSSETSCDCSEPIYIRGCQPKIYDGKIFPGKGGEKQWICKDTIIHGDTNGACIPPRTQNLCVGELWDKRYGGRSNIKNDTKESLKQKIKNAIQKETELLYEYHDKGTAIISRNPMKGQKEKEEKNNDSNGLPKGFCHAVQRSFIDYKNMILGTSVNIYEYIGKLQEDIKKIIEKGTTKQNGKTVGSGAENVNAWWKGIEGEMWDAVRCAITKINKKQKKNGTFSIDECGIFPPTGNDEDQSVSWFKEWSEQFCIERLQYEKNIRDACTNNGQGDKIQGDCKRKCEEYKKYISEKKQEWDKQKTKYENKYVGKSASDLLKENYPECISANFDFIFNDNIEYKTYYPYGDYSSICSCEQVKYYEYNNAEKKNNKSLCHEKGNDRTWSKKYIKKLENGRTLEGVYVPPRRQQLCLYELFPIIIKNKNDITNAKKELLETLQIVAEREAYYLWKQYHAHNDTTYLAHKKACCAIRGSFYDLEDIIKGNDLVHDEYTKYIDSKLNEIFDSSNKNDIETKRARTDWWENEAIAVPNITGANKSDPKTIRQLVWDAMQSGVRKAIDEEKEKKKPNENFPPCMGVQHIGIAKPQFIRWLEEVVTNEFCEKYTKYFEDMKSNCNLRKGADDCDDNSNIECKKACANYTNWLNPKRIEWNGMSNYYNKIYRKSNKESEDGKDYSMIMEPTVIDYLNKRCNGEINGNYICCSCKNIGENSTSGTVNKKLQKKETQCEDNKGPLDLMNKVLNKMDPKYSEHKMKCTEVYLEHVEEQLKEIDNAIKDYKLYPLDRCFDDKSKMKVCDLIGDAIGCKHKTKLDELDEWNDVDMRDPYNKYKGVLIPPRRRQLCFSRIVRGPANLRNLKEFKEEILKGAQSEGKFLGNYYNEDKDKEKALEAMKNSFYDYEYIIKGSDMLTNIQFKDIKRKLDRLLEKETNNTEKVDDWWETNKKSIWNAMLCGYKKSGNKIIDPSWCTIPTTETPPQFLRWIKEWGTNVCIQKEEHKEYVKSKCSNVTNLGAQESESKNCTSEIKKYQEWSRKRSIQWEAISEGYKKYKGMDEFKNTFKNIKEPDANEPNANEYLKKHCSKCPCGFNDMQEITKYTNIGNEAFKQIKEQVDIPAELEDVIYRLKHHEYDKGNDYICNKYKNINVNMKKNNDDTWTDLVKNSSDINKGVLLPPRRKNLFLKIDESDICKYKRDPKLFKDFIYSSAISEVERLKKVYGEAKTKVVHAMKYSFADIGSIIKGDDMMENNSSDKIGKILGDGVGQNEKRKKWWDMNKYHIWESMLCGYKHAYGNISENDRKMLDIPNNDDEHQFLRWFQEWTENFCTKRNELYENMVTACNSAKCNTSNGSVDKKECTEACKNYSNFILIKKKEYQSLNSQYDMNYKETKAEKKESPEYFKDKCNGECSCLSEYFKDETRWKNPYETLDDTEVKNNCMCKPPPPASNNTSDILQKTIPFGIALALGSIAFLFMKKKPKTPVDLLRVLDIPKGDYGIPTPKSSNRYIPYASDRYKGKTYIYMEGDTSGDDDKYIWDL >FCR3 complete 2734 aa extracellular part (577 aa highlighted corr. ID1-DBL2b) (SEQ ID NO: 56)MDSTSTIANKIEEYLGAKSDDSKIDELLKADPSEVEYYRSGGDGDYLKNNICKITVNHSDSGKYDPCEKKLPPYDDNDQWKCQQNSSDGSGKPENICVPPRRERLCTYNLENLKFDKIRDNNAFLADVLLTARNEGEKIVQNHPDTNSSNVCNALERSFADLADIIRGTDQWKGTNSNLEKNLKQMFAKIRENDKVLQDKYPKDQKYTKLREAWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSDRKKNFELCRKCGHYEKEVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTREDHKSKEGTSYCSTCKDKCKKYCECVKKWKTEWENQENKYKDLYEQNKNKTSQKNTSRYDDYVKDFFEKLEANYSSLENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDGNDVTFFNLFEQWNKEIQYQIEQYMTNANISCIDEKEVLDSVSDEGTPKVRGGYEDGRNNNTDQGTNCKEKCKCYKLWIEKINDQWGKQKDNYNKFRSKQIYDANKGSQNKKVVSLSNFLFFSCWEEYIQKYFNGDWSKIKNIGSDTFEFLIKKCGNNSAHGEEIFNEKLKNAEKKCKENESTDTNINKSETSCDLNATNYIRGCQSKTYDGKIFPGKGGEKQWICKDTIIHGDTNGACIPPRTQNLCVGELWDKSYGGRSNIKNDTKELLKEKIKNAIHKETELLYEYHDTGTAIISKNDKKGQKGKNDPNGLPKGFCHAVQRSFIDYKNMILGTSVNIYEHIGKLQEDIKKIIEKGTPQQKDKIGGVGSSTENVNAWWKGIEREMWDAVRCAITKINKKNNNSIFNGDECGVSPPTGNDEDQSVSWFKEWGEQFCIERLRYEQNIREACTINGKNEKKCINSKSGQGDKIQGACKRKCEKYKKYISEKKQEWDKQKTKYENKYVGKSASDLLKENYPECISANFDFIFNDNIEYKTYYPYGDYSSICSCEQVKYYKYNNAEKKNNKSLCYEKDNDMTWSKKYIKKLENGRSLEGVYVPPRRQQLCLYELFPIIIKNEEGMEKAKEELLETLQIVAEREAYYLWKQYNPTGKGIDDANKKACCAIRGSFYDLEDIIKGNDLVHDEYTKYIDSKLNEIFGSSDTNDIDTKRARTDWWENETITNGTDRKTIRQLVWDAMQSGVRYAVEEKNENFPLCMGVEHIGIAKPQFIRWLEEVVTNEFCEKYTKYFEDMKSKCDPPKRADTCGDNSNIECKKACANYTNWLNPKRIEWNGMSNYYNKIYRKSNKESEGGKDYSMIMAPTVIDYLNKRCHGEINGNYICCSCKNIGAYNTTSGTVNKKLQKKETECEEEKGPLDLMNEVLNKMDKKYSAHKMKCTEVYLEHVEEQLNEIDNAIKDYKLYPLDRCFDDQTKMKVCDLIADAIGCKDKTKLDELDEWNDMDLRGTYNKHKGVLIPPRRRQLCFSRIVRGPANLRSLNEFKEEILKGAQSEGKFLGNYYKEHKDKEKALEAMKNSFYDYEDIIKGTDMLTNIEFKDIKIKLDRLLEKETNNTKKAEDWWKTNKKSIWNAMLCGYKKSGNKIIDPSWCTIPTTETPPQFLRWIKEWGTNVCIQKQEHKEYVKSKCSNVTNLGAQASESNNCTSEIKKYQEWSRKRSIRWETISKRYKKYKRMDILKDVKEPDANTYLREHCSKCPCGFNDMEEMNNNEDNEKEAFKQIKEQVKIPAELEDVIYRIKHHEYDKGNDYICNKYKNIHDRMKKNNGNFVTDNFVKKSWEISNGVLIPPRRKNLFLYIDPSKICEYKKDPKLFKDFIYWSAFTEVERLKKAYGGARAKVVHAMKYSFTDIGSIIKGDDMMEKNSSDKIGKILGDTDGQNEKRKKWWDMNKYHIWESMLCGYREAEGDTETNENCRFPDIESVPQFLRWFQEWSENFCDRRQKLYDKLNSECISAECTNGSVDNSKCTHACVNYKNYILTKKTEYEIQTNKYDNEFKNKNSNDKDAPDYLKEKCNDNKCECLNKHIDDKNKTWKNPYETLEDTFKSKCDCPKPLPSPIKPDDLPPQADEPFDPTILQTTIPFGIALALGSIAFLFMKVIYIYIYVCCICMYVCMYVCMYVCMYVCMYVCMHVCMLCVYVIYVFKICIYIEKEKRKK >BPTI, protease inhibitor  (SEQ ID NO: 57)RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA>PE38, Pseudomonas exotoxin A, (underlining of KDEL represent a signalsequence, which may be optional for the constructs according to the present invention) (SEQ ID NO: 58)RH RQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL>PE38LR, variant of PE38 (underlining of KDEL represent a signal sequence,which may be optional for the constructs according to the present invention)(SEQ ID NO: 59)RH RQPRGWEQLYPTGAEFLGDGGDISFSTRGTQNVVTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDELSequences of VAR2CSA polypeptides fused with truncated fragments of Pseudomonas exotoxin A (PE38)Fused VAR2CSA-PE38 proteins may have modifications such as a protease inhibitor (BPTI) in the N-terminal and/or an optimized PE38 sequence that is less immunogenic (PE38LR) >BPTI-ID1-ID2aFCR3-PE38LR, underlined sequence corresponds to the ID1domain of FCR3, sequence in bold corresponds to DBL2Xb domain of FCR3, underlined and bold sequence is ID2a (SEQ ID NO: 60)RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGANYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACOCKIPTNEETCDDRKEYMNOWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLD RHRQPRGWEQLYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >BPTI-ID1-ID2aFCR3-PE38  (SEQ ID NO: 61)RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGANYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNVVTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >ID1-ID2aFCR3-PE38 (SEQ ID NO: 62)NYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPVVTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >ID1-ID2aFCR3-PE38LR  (SEQ ID NO: 63)NYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPVVTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDRHRQPRGWEQLYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >BPTI-DBL1-ID2aFCR3-PE38LR (SEQ ID NO: 64)RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGANHSDSGKYDPCEKKLPPYDDNDQWKCQQNSSDGSGKPENICVPPRRERLCTYNLENLKFDKIRDNNAFLADVLLTARNEGEKIVQNHPDTNSSNVCNALERSFADLADIIRGTDQWKGTNSNLEKNLKQMFAKIRENDKVLQDKYPKDQKYTKLREAWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSDRKKNFELCRKCGHYEKEVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTREDHKSKEGTSYCSTCKDKCKKYCECVKKWKTEWENQENKYKDLYEQNKNKTSQKNTSRYDDYVKDFFEKLEANYSSLENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIVVTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPVVTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDRHRQPRGWEQLYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >BPTI-DBL1-ID2aFCR3-PE38  (SEQ ID NO: 65)RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGANHSDSGKYDPCEKKLPPYDDNDQWKCQQNSSDGSGKPENICVPPRRERLCTYNLENLKFDKIRDNNAFLADVLLTARNEGEKIVQNHPDTNSSNVCNALERSFADLADIIRGTDQWKGTNSNLEKNLKQMFAKIRENDKVLQDKYPKDQKYTKLREAWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSDRKKNFELCRKCGHYEKEVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTREDHKSKEGTSYCSTCKDKCKKYCECVKKWKTEWENQENKYKDLYEQNKNKTSQKNTSRYDDYVKDFFEKLEANYSSLENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIVVTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPVVTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAAnGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTsLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >DBL1-ID2aFCR3-PE38LR (SEQ ID NO: 66)NHSDSGKYDPCEKKLPPYDDNDQWKCQQNSSDGSGKPENICVPPRRERLCTYNLENLKFDKIRDNNAFLADVLLTARNEGEKIVQNHPDTNSSNVCNALERSFADLADIIRGTDQWKGTNSNLEKNLKQMFAKIRENDKVLQDKYPKDQKYTKLREAWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSDRKKNFELCRKCGHYEKEVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTREDHKSKEGTSYCSTCKDKCKKYCECVKKWKTEWENQENKYKDLYEQNKNKTSQKNTSRYDDYVKDFFEKLEANYSSLENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIVVTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDRHRQPRGWEQLYPTGAEFLGDGGDISFSTRGTQNVVTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >DBL1-ID2aFCR3-PE38  (SEQ ID NO: 67)NHSDSGKYDPCEKKLPPYDDNDQWKCQQNSSDGSGKPENICVPPRRERLCTYNLENLKFDKIRDNNAFLADVLLTARNEGEKIVQNHPDTNSSNVCNALERSFADLADIIRGTDQWKGTNSNLEKNLKQMFAKIRENDKVLQDKYPKDQKYTKLREAWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSDRKKNFELCRKCGHYEKEVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTREDHKSKEGTSYCSTCKDKCKKYCECVKKWKTEWENQENKYKDLYEQNKNKTSQKNTSRYDDYVKDFFEKLEANYSSLENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIVVTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >ID1-ID2a3D7-PE38 (SEQ ID NO: 68)LSFILNSSDANNPSEKIQKNNDEVCNCNESGIASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADKAPVVTTYTTYTTTEKCNKETDKSKLQQCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNVVTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >ID1-ID2a3D7-PE38LR (SEQ ID NO: 69)LSFILNSSDANNPSEKIQKNNDEVCNCNESGIASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADKAPVVTTYTTYTTTEKCNKETDKSKLQQCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDRHRQPRGWEQLYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >ID1-DBL2IDFCR3-PE38LR  (SEQ ID NO: 70)NYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPVVTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYRHRQPRGWEQLYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPRKDEL >DT388, sequence of diphtheria toxin (SEQ ID NO: 71)MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPMHEFSequences of VAR2CSA polypeptides fused with truncated fragments of diphtheria toxin >DT388-DBL1-ID2a 3D7  (SEQ ID NO: 72)MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPMHEFHSDSGTNDPCDRIPPPYGDNDQWKCAIILSKVSEKPENVFVPPRRQRMCINNLEKLNVDKIRDKHAFLADVLLTARNEGERIVQNHPDTNSSNVCNALERSFADIADIIRGTDLWKGTNSNLEQNLKQMFAKIRENDKVLQDKYPKDQNYRKLREDWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSNGDNKLELCRKCGHYEEKVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTSEDHKSKEGTSYCSTCKDKCKKYCECVKKWKSEWENQKNKYTELYQQNKNETSQKNTSRYDDYVKDFFKKLEANYSSLENYIKGDPYFAEYATKLSFILNSSDANNPSEKIQKNNDEVCNCNESGIASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQQCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDSGR >DT388-DBL1-ID2a FCR3 (SEQ ID NO: 73)MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPMHEFHSDSGKYDPCEKKLPPYDDNDQWKCQQNSSDGSGKPENICVPPRRERLCTYNLENLKFDKIRDNNAFLADVLLTARNEGEKIVQNHPDTNSSNVCNALERSFADLADIIRGTDQWKGTNSNLEKNLKQMFAKIRENDKVLQDKYPKDQKYTKLREAWWNANRQKVWEVITCGARSNDLLIKRGWRTSGKSDRKKNFELCRKCGHYEKEVPTKLDYVPQFLRWLTEWIEDFYREKQNLIDDMERHREECTREDHKSKEGTSYCSTCKDKCKKYCECVKKWKTEWENQENKYKDLYEQNKNKTSQKNTSRYDDYVKDFFEKLEANYSSLENYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDSGR >DT388-ID1-ID2a 3D7 (SEQ ID NO: 74)MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPMHEFLSFILNSSDANNPSEKIQKNNDEVCNCNESGIASVEQEQISDPSSNKTCITHSSIKANKKKVCKHVKLGVRENDKDLRVCVIEHTSLSGVENCCCQDFLRILQENCSDNKSGSSSNGSCNNKNQEACEKNLEKVLASLTNCYKCDKCKSEQSKKNNKNWIWKKSSGKEGGLQKEYANTIGLPPRTQSLCLVVCLDEKGKKTQELKNIRTNSELLKEWIIAAFHEGKNLKPSHEKKNDDNGKKLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQKIFGKLFRKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIWLAMKHGAGMNSTTCCGDGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVEHFCKQRQEKVKPVIENCKSCKESGGTCNGECKTECKNKCEVYKKFIEDCKGGDGTAGSSWVKRWDQIYKRYSKYIEDAKRNRKAGTKNCGPSSTTNAAENKCVQSDIDSFFKHLIDIGLTTPSSYLSIVLDDNICGADKAPWTTYTTYTTTEKCNKETDKSKLQQCNTAVVVNVPSPLGNTPHGYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDSGR >DT388-ID1-ID2a FCR3 (SEQ ID NO: 75)MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPMHEFNYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCDKCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLDELRESWWNTNKKYIVVTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSSTTNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPVVTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLDSGR

EXAMPLES Example 1 Production of Truncated Recombinant VAR2CSA Proteins

All protein truncations were produced according to previously defineddomain borders (Dahlbäck M, Jorgensen L M, Nielsen M A, Clausen T M,Ditlev S B, et al. J Biol Chem 286: 15908-15917). For the purpose ofsimplification we have divided the CIDR_(PAM) domain into two domainsID2a and ID2b, where ID2a is the N-terminal part of CIDR_(PAM) notcontaining the CIDR-like sequence and ID2b corresponds to the CIDR-likesequence. We also used a new DBL2X border incorporating 93 amino acidsof ID2a. For simplification we call this border DBL2Xb, while the oldborder will be referred to as DBL2Xa. Primers used in cloning are listedin Table 2. Fragments were expressed in baculovirus-infected insectcells as soluble proteins as described in Method 1. Most proteins wereproduced based on the FCR3 genotype. Some FCR3 fragments did not expressand these were instead made based on the 3D7 genotype. The proteins wereused interchangeably in the analysis since we show that recombinantVAR2CSA from both genotypes bind equally to CSA. All proteins showed ashift in gel mobility when comparing reduced and non-reduced samples bySDS-PAGE (Method 2). This is consistent with the formation ofintra-molecular disulfide bridges. Some proteins formed high-molecularweight complexes detected by non-reduced SDS-PAGE. This is probably dueto the formation of inter-molecular disulfide bridges between unpairedcysteines. This was confirmed by reducing the complexes to monomericprotein using DTT.

TABLE 2 Cloning Primers FCR3 Primers Protein Forward PrimerReverse Primer ID1-ID2b AACTACATCAAGGGCGAC (SEQ ID NO: 76)CTTGTTGATATTGGTGTCGGT (SEQ ID NO: 77) DBL1X-ID2aCACAGCGATAGCGGCAAG (SEQ ID NO: 78) GTCCAGCTTGCTGGAGTT (SEQ ID NO: 79)ID1-ID2a AACTACATCAAGGGCGAC (SEQ ID NO: 80)GTCCAGCTTGCTGGAGTT (SEQ ID NO: 81) ID1-DBL2XaAACTACATCAAGGGCGAC (SEQ ID NO: 82) AGCGGCGTTGGTGGTGGA (SEQ ID NO: 83)ID1-DBL2Xb AACTACATCAAGGGCGAC (SEQ ID NO: 84)GTACTTGTACCGGTAGGG (SEQ ID NO: 85) DBL1X-DBL2XbCACAGCGATAGCGGCAAG (SEQ ID NO: 86) GTACTTGTACCGGTAGGG (SEQ ID NO: 87)3d7 Primers Protein Forward Primer Reverse Primer DBL2X-DBL4ECTGACCAACTGCTACAAG (SEQ ID NO: 88) GGTCCAGAGGGTACAGCTT (SEQ ID NO: 89)ID1-DBL3E CTGTCCTTCATCCTGAAC (SEQ ID NO: 90)TTCAGCGTTGTTGTACTCGTA (SEQ ID NO: 91) ID1-DBL4ECTGTCCTTCATCCTGAAC (SEQ ID NO: 92) GTCCAGAGGGTACAGCTT (SEQ ID NO: 93)DBL1X-ID2b CACTCTGACTCTGGCACC (SEQ ID NO: 94)AGAGGACTTCATCTTGTTGTTGGT (SEQ ID NO: 95) ID1-ID2bCTGTCCTTCATCCTGAAC (SEQ ID NO: 96)AGAGGACTTCATCTTGTTGTTGGT (SEQ ID NO: 97) DBL1X-ID2aCACTCTGACTCTGGCACC (SEQ ID NO: 98) GTCCAGCTTAGAGGAGTT (SEQ ID NO: 99)ID1-ID2a CTGTCCTTCATCCTGAAC (SEQ ID NO: 100)GTCCAGCTTAGAGGAGTT (SEQ ID NO: 101) DBL1X-DBL2XaCACTCTGACTCTGGCACC (SEQ ID NO: 102) GGCGGCGTTGGTGGTAGA (SEQ ID NO: 103)ID1-DBL2Xa CTGTCCTTCATCCTGAAC (SEQ ID NO: 104)GGCGGCGTTGGTGGTAGA (SEQ ID NO: 105) DBL1X-DBL2XbCACTCTGACTCTGGCACC (SEQ ID NO: 106) GTACTTGTATCCGTGGGG (SEQ ID NO: 107)ID1-DBL2Xb CTGTCCTTCATCCTGAAC (SEQ ID NO: 108)GTACTTGTATCCGTGGGG (SEQ ID NO: 109) Mutating Putative CSA Binding SitesPCR1 Fragment 1 Protein Forward Reverse DBL1X-ID2a (DSMCACAGCGATAGCGGCAAG (SEQ ID NO: 110) GGTGTCGAAGTTGATGTCGGGCAGATTGCCCAGGTADeletion) (SEQ ID NO: 111) Alanine sub.CACAGCGATAGCGGCAAG (SEQ ID NO: 112) AGCTGCGGCCAGATTAGCGCCCTCGTGGAAGGACACK(626,629,630), R(631) (SEQ ID NO: 113) Alanine sub.CACAGCGATAGCGGCAAG (SEQ ID NO:114) AGCGCATTCAGCTGCGGCGTTGGTCTTGATGGAGCTK(459,460,461,464) (SEQ ID NO: 115) Fragment 2 Protein Forward ReverseDBL1X-ID2a (DSM CACAGCGATAGCGGCAAG (SEQ ID NO: 116)GTCCAGCTTGCTGGAGTT (SEQ ID NO: 117) Deletion) Alanine sub.GCTAATCTGGCCGCAGCTTACCCCCAGAATAAGAAC GTCCAGCTTGCTGGAGTT (SEQ ID NO: 119)K(626,629,630), R(631) (SEQ ID NO: 118) Alanine sub.GCCGCAGCTGAATGCGCTGACGTGAAGCTGGGCGTG GTCCAGCTTGCTGGAGTT (SEQ ID NO: 121)K(459,460,461,464) (SEQ ID NO: 120) PCR2 Final Constuct Protein ForwardReverse DBL1X-ID2a (DSM CACAGCGATAGCGGCAAG (SEQ ID NO: 122)GTCCAGCTTGCTGGAGTT (SEQ ID NO: 123) Deletion) Alanine sub.CACAGCGATAGCGGCAAG (SEQ ID NO: 124) GTCCAGCTTGCTGGAGTT (SEQ ID NO: 125)K(626,629,630), R(631) Alanine sub. CACAGCGATAGCGGCAAG (SEQ ID NO: 126)GTCCAGCTTGCTGGAGTT (SEQ ID NO: 127) K(459,460,461,464)

Example 2 VAR2CSA from FCR3 and 3D7 Binds CSA with Similar Affinity andSpecificity

FCR3 infected erythrocytes (IE) adhere much stronger to CSA in vitrocompared to 3D7 or NF54 (IE). If the differences are related to thesequence differences of the expressed VAR2CSA the information could beused to define residues involved in the adhesion process. To test this,we produced a series of overlapping 3D7 VAR2CSA fragments, identical tothe ones we have previously tested for FCR3.

The proteins were first screened for specific CSPG binding in a solidphase binding assay (ELISA) (described in Method 3). Proteins bindingspecifically to CSPG were then further purified by size exclusionchromatography, to obtain pure monomeric protein, and subjected tokinetic analysis on a Quartz Crystal Microbalance (Attana A100) (Method2 and 4, respectively). The 3D7 VAR2CSA fragments showed bindingcharacteristics very similar to their FCR3 counterparts in the solidstate binding assay. The same is true in the kinetic analysis (Table 3).The sensorgrams show association and dissociation data collected atdifferent protein concentrations. This allows determination of theassociation rate constant (k_(on)), disassociation rate constant(k_(off)), and the equilibrium constant (K_(D)). Together with the peakresponse levels these parameters give estimation for the CSPG bindingaffinity. There is no apparent difference between 3D7 and FCR3fragments. Some fragments show lower affinity, but this characteristicis maintained in the fragments counterpart. This indicates that the 3D7and FCR3 VAR2CSA proteins fold and function in the same way.

TABLE 3 CSA binding affinity of produced VAR2CSA proteins. Affinity isgiven as a K_(D) (nM) value determined in kinetics experiments using aquartz crystal microbalance biosensor (Attana A100). FCR3 3D7 VAR2CSAFragment Baculo E. coli Baculo FV2 5.2* 8.2 ID1-DBL4ε 8.6* 9.4 ID1-DBL3ε0.3* 8.5 DBL2X-DBL4ε 2.4* 1.2 DBL1-ID2b 1.5* DBL1-ID2a 8.0 3.5 29.5ID1-ID2a 7.6 18.3 5.7 DBL1X-DBL2Xb 14.6 DBL1X-DBL2Xa N/A ID1-DBL2Xb 21.8ID1-DBL2Xa N/A *Proteins published in (Dahlbäck et al, JBC, 2011) N/A:proteins for which no K_(D) value could be determined, due to a lack ofbinding to CSA.

Example 3 The Core-CSA Binding Site Lies within the DBL2X Domain

It has been suggested that the minimal CSA binding region in VAR2CSAlies within DBL2X-ID2b, with the need of flanking domains for fullaffinity binding (Dahlbäck M, Jorgensen L M, Nielsen M A, Clausen T M,Ditlev S B, et al. J Biol Chem 286: 15908-15917). Here we have analyzedshorter fragments of VAR2CSA to further map the regions required for CSAbinding.

The truncated proteins were first screened for binding to a CSAproteoglycan (CSPG) in ELISA and then further purified to obtainmonomers for examination on the Quartz Crystal Microbalance (Methods 3,2 and 4, respectively). The minimal binding region is ID1-DBL2Xb (Table3). This region showed a binding affinity of 21.8 nM, which iscomparable to that of full-length VAR2CSA.

Placental IEs are highly selective for low-sulfated placental CSPG. Theydo not adhere to any other glycosaminoglycans (GAG), such as heparansulfate (HS). The same is true for the full-length recombinant VAR2CSAprotein. The solid state binding assay showed that the VAR2CSAfragments, containing the minimal CSA binding region, bound specificallyto CSA. To confirm this the minimal binding fragments were furthertested for binding to a heparan sulfate proteoglycan (HSPG) on theQuartz Crystal Microbalance (Method 4). None of the fragments boundHSPG.

Example 4 Antibodies Induced Against Novel Minimum Binding RegionsInduce a Potent Parasite Anti-Adhesive Immune Response

A VAR2CSA based vaccine against PM must be able to induce a strongprotective immune response. In this, the most important aspect is theformation of anti-VAR2CSA IgG antibodies capable of inhibiting placentalsequestration. We have examined the molecular mechanism underlying theVAR2CSA-CSA interaction for the purpose of designing optimal vaccineantigens. To test whether our produced VAR2CSA recombinant fragmentsshowed the capacity to induce an adhesion blocking immune response, theywere used in rat immunizations (Method 6).

VAR2CSA fragment-specific serum was tested for ability to inhibit IEadhesion to CSPG (Method 11). Antibodies raised against all CSA-bindingfragments were very potent inhibitors of binding. In fact binding wasinhibited nearly 100% in all cases. DBL1X-DBL2Xa and ID1-DBL2Xa were notgood inhibitors, consistent with the lack of CSA binding of thesefragments (Table 3). The data implies that the CSA-binding proteins areproperly folded and support the localization of the above-definedminimal binding region.

Example 5 Epitopes Responsible for the Induction of Anti-AdhesiveAntibodies Lies within the Minimal Binding Region

To examine if the inhibitory anti-FV2 response is directed towards theminimal binding region, we affinity purified FV2 antibodies on four ofthe previous described VAR2CSA fragments (Method 7). Thefragment-specific antibodies were then tested for the capacity toinhibit VAR2CSA expressing parasite binding to CSPG (Method 11).Antibodies purified on immobilized ID1-DBL4s, DBL1X-ID2a and ID1-ID2a,fully inhibited parasite adhesion. Furthermore, the depleted FV2 sampleslost a significant portion of their inhibitory capacity. This indicatesthat epitopes inducing anti-adhesive antibodies are present within thesefragments. Antibodies purified on DBL1X-DBL2Xa show a reduced inhibitorycapacity, consistent with the lack of CSA binding of this fragment(Table 3). The data suggest that epitopes responsible for induction ofinhibitory antibodies are located within the minimal binding region(here illustrated by ID1-ID2a).

Example 6 Mutating Putative GAG Binding Sites in the Minimal BindingRegion has No Effect on CSPG Binding

Characterizing the nature of the interaction between VAR2CSA and CSA isimportant for the design of multivalent PM vaccines. In this, a majorpart is identification of the specific CSA-binding site andcharacterization of the underlying chemical interactions. Sequenceanalysis of the minimal CSA-binding region revealed two conservedputative GAG binding sites. One is located in the ID1 region and has theclassic Cardin-Weintraub XBBBXXBX motif (Cardin, A. D., and Weintraub,H. J. (1989) Arteriosclerosis 9, 21-32) (458-NKKKECKD-465). Another, inDBL2X, has the same motif in reverse (625-GKNLKKRY-632). It has alsobeen hypothesized that a dimorphic sequence motif (DSM), found in theN-terminal part of DBL2X, is involved in binding CSA (Sander, A. F.,Salanti, A., Laystsen, T., Nielsen, M. A., Magistrado, P., Lusingu, J.,Ndam, N. T., and Arnot, D. E. (2009) PLoS One 4, e6667). To test whetherthese putative sites had a function in CSA binding, we substituted basicamino acids in the classic GAG binding sites with alanines and made aten amino acid (590-KLENVCEDVK-603) deletion in the middle of a surfaceexposed loop within the DSM region. All mutations were performed in theDBL1X-ID2a fragment.

Substituting basic amino acids in the putative ID1 and DBL2X GAG bindingsites, with alanines had no effect on CSPG binding. No decrease in CSPGbinding was seen compared to the wild-type protein in ELISA (Method 3).The construct with four alanine substitutions, Alanine Sub. K(459, 460,461, 464), shows considerable HSPG binding, which could be caused by achange in protein structure in response to mutation. The two mutants,Alanine Sub. K(626, 629, 630), R(631) and Alanine Sub. K(459, 460, 461,464), show CSPG binding kinetics similar to the positive control (Method4). This is evident by similar K_(D) values and peak responses.

The deletion of the DSM region did not reduce binding to CSPG (Methods 3and 4). The DSM knock out mutant shows considerable binding to HSPG inELISA. This is likely caused by an erroneous cloning where 100 aminoacids of DBL1X were lost. Importantly CSPG binding was not affected.

Example 7 VAR2CSA Binding to CSPG does not Depend on Ionic Interactions

Mutation of the classic Cardin-Weintraub GAG binding motifs had noeffect on CSPG binding. This indicates that the VAR2CSA-CSA bindingmechanism differs from the general mode of sulfate binding in classicGAG binding models. There are examples of GAG binding proteins showinglittle dependence on ionic interactions with the sulfated GAG structure.To test if this was the case, we examined ionic dependence according tothe polyelectrolyte theory (Record, M. T., Jr., Lohman, M. L., and DeHaseth, P. (1976) J Mol Biol 107, 145-158).

Glycosaminoglycans, like DNA, are highly charged polymers often referredto as polyelectrolytes. The negatively charged groups incur a highdegree of repulsive energy within each polymer. Monovalent cations, suchas Na⁺, interact with the negatively charged groups to minimize therepulsive energy. Binding of basic amino acids to the sulfate groupsdisplaces the bound cations and leads to the release of free energy. Thefavorable release of bound Na⁺ ions is referred to as thepolyelectrolyte effect.

The theory states that the binding of a protein to a GAG can bedescribed by:

Protein+GAG(m sites)

Protein−GAG+m(1−f)Na⁺

Where m is the number of Na⁺ ions released upon binding of a singleprotein and f is the fraction of anions not shielded by Na⁺ ions.According to the theory the observed K_(D) value is related to ionic andnon-ionic contributions by:

Log K_(D,observed)=Log K_(D,nonionic) +m(1−f)Log [Na⁺]

Where K_(D,nonionic) is the disassociation constant in the absence ofionic interactions. A plot of Log K_(D,observed), vs Log [Na⁺] is linearwith a slope of m(1−f). Thus, if the fraction of unshielded anions (f)is known, the number of ionic interactions involved in the binding canbe determined. For heparin (1−f) is 0.8 (Olson, S. T., Halvorson, H. R.,and Bjork, I. (1991) J Biol Chem 266, 6342-6352). The value is not knownfor CSA, but (1-f) cannot exceed 1. We can therefore estimate themaximal number of ionic interactions involved. Furthermore, when [Na⁺]=1M, Log [Na⁺]=0, which means that at this Na⁺ concentration LogK_(D,observed)=Log K_(D,nonionic).

We tested the binding of FV2, DBL1X-ID2a and ID1-ID2a to CSPG in a solidstate binding assay at different concentrations of NaCl (150 mM, 200 mM,250 mM, 300 mM), by performing titrations of binding from 400 nM-1.65 nMprotein in a 1:2 dilution series (Method 5). The observed K_(D) valueswere determined as the protein concentration giving half-maximum(B_(max)) response. This was done using non-linear regression (leastsquares fit with Hill slope) in Graphpad Prism. Higher saltconcentrations were not included in the analysis as binding was almostcompletely inhibited. This is probably due to a change in proteinstructure. This notion is supported by the fact that Log K_(D,observed)vs Log [Na⁺] was linear only between 150 mM and 300 mM, suggesting thatother factors play a role at higher concentrations of NaCl.

Log K_(D,observed) vs Log [Na⁺] shows a linear relationship. The slopem(1−f) ranges between 2.7 for ID1-ID2a and 3.4 for full-length (FV2). Wedo not know the value for f, but the maximal number of ionicinteractions involved in the binding must be between 2 and 3. It isinteresting that the value for the full-length protein is higher thanfor the short fragments, indicating that this protein makes an extraionic interaction with CSPG. The K_(D) values at 150 mM NaCl serves asour reference point, as this is the physiological NaCl concentration. Byextrapolating the linear relationship and finding the y-intercept wefind that K_(D,nonionic)=5.9 μM for FV2, K_(D,nonionic)=3.4 μM forDBL1X-ID2a, and K_(D,nonionic)=0.7 μM for ID1-ID2a. Comparing thelogarithmic values of these and the reference point (150 mM NaCl), weestimate that between 25-35% of the VAR2CSA binding can be accounted forby ionic interactions. This suggests that the high CSA affinity forVAR2CSA cannot be explained by ionic interactions with the sulfated GAGstructure alone. The high affinity may be achieved through a complexbinding site making a multivalent interaction with the CSA carbohydratebackbone.

Example 8 VAR2CSA Minimal CSA Binding Region Binds Specifically to aWide Panel of Cancer Cells

Many different cancer cells have been associated with high expression ofthe proteoglycan CSPG4. This molecule was initially described as amarker for melanoma but it has recently been found in many cancer forms,including cancer stem cells. The CS chain(s) attached to CSPG4 is knownto be primarily CSA. One of the smallest VAR2CSA fragments (ID1-ID2a)was analyzed for binding to a large panel of various cancer cell linesby flow-cytometry (Method 12a and 12b). The non-CSA binding proteinID1-DBL2Xa was used as a negative control. The VAR2CSA recombinantprotein (ID1-ID2a) binds strongly at 75 nM to all cancer cell linestranscribing CSPG4 (microarray data) including cutaneous Melanoma (C32,MeWo), Lung carcinoma (A549), Breast carcinoma (HCC1395), Osterosarcoma(U20S, MNNG/HOS), Rhabdomyosarcoma (RH30) (Table 4 and 5). This proteinalso binds strongly to cutaneous T-cell lymphoma, which does not expressCSPG4 (Table 4). The negative control protein ID1-DBL2Xa did not bind toany of the cell lines tested (Table 4). In addition, ID1-ID2a did notinteract with human red blood cells, which were used as control cells.Wild type and GAG-deficient Chinese hamster ovary (CHO) cells were alsoanalyzed for ID1-ID2a interaction. The strong interaction seen forID1-ID2a with wild-type CHO cells was completely abolished whenanalyzing the CHO-745 cell line, in which the GAG-synthesis isdisrupted. The CSA specificity of the interaction was also verified byinhibiting VAR2CSA binding to cells by pre-mixing VAR2CSA with CSA, CSCor HS. CSC and HS did not have any effect on the binding, whereas CSAefficiently abrogated binding of VAR2CSA to the cancer cells.

Following these results, a larger panel of cancer cells were screened byflow cytometry (Table 6 and 7) using the DBL1-ID2a or ID1-ID2a fragmentof VAR2CSA. The main purpose of this screening is to identify cell linessuitable for xenograft modeling in vivo.

TABLE 4 Staining of cancer cell lines and negative control cells usingthe minimal binding domain of VAR2CSA (ID1-ID2a). Cells were incubatedwith medium alone (blank) or recombinant proteins (ID1-DBL2 or ID1-ID2a)at 75 nM for 30 minutes, followed by incubation with anti-V5-FITC(Invitrogen) at 1:800, cells were washed thrice between each incubation.Shown are the mean FITC fluorescence values recorded from a minimum of5000 cells using a FC500 flowcytometer (Becton Dickinson). Cell typeBlank ID1-DBL2Xa ID1-ID2a C32 5.77 6.94 63.81 MyLa 2059 5.61 5.61 145.35MyLa 1850 5.87 5.6 137.86 Cho WT 3.09 4.35 34.79 Cho 745 4.24 4.29 4.38PBMC 1.34 1.36 1.67 Erythrocytes 1.11 1.17 1.07

TABLE 5 Staining of cancer cell lines using recombinant VAR2CSA Cellswere incubated with medium alone (blank) or recombinant proteins(DBL1-ID2a or ID1-ID2a) at 75 nM for 30 minutes, followed by incubationwith anti-V5-FITC (Invitrogen) at 1:800, cells were washed thricebetween each incubation. Shown are the medium score of FITC fluorescenceintensity recorded from a minimum of 4 high power field images using aHAL100 Zeiss microscope. Cell type Blank DBL1-ID2a U2OS NS +++ MG63 NS++++ MDA-MB-231 NS +++ TC32 NS + TC71 NS ++ MNNG NS +++ CHLA9 NS ++CHLA10 NS ++ RH30 NS +++ RH18 NS ++ PC3 NS +++ NS: No staining; +: weak;++: medium; +++: strong; ++++: Very strong.

TABLE 6 Screening of diverse human cancer cell lines for binding ofrecombinant VAR2CSA (using DBL1-ID2a or ID1-ID2a). Binding was measuredby flow cytometry as described in METHOD 12. 75 nM 150 nM Cell lineControl VAR2CSA VAR2CSA Comments MeWo NS +++ ++++ Melanoma (Fibroblastmorphology, derived from lymphnode) A549 NS +++ +++ Lung Adenocarcinoma(K-RasG12S) HCC1395 NS +++ ++++ Invasive ductal breast carcinoma TNMstage 1 grade 3; no lymphnode metastasis; Her2-neg, ER-neg, PR-neg(Triple-negative) RH30 NS +++ ++++ Rhabdomyosarcoma (TPp53 negativ;PAX7-FOXO1A fusion positive; highly genomic instable (>50 chromosomerearangements)) MNNG NS +++ +++ Osteosarcoma from 13 year old femalecaucasian (TPR-Met positive) U2OS NS +++ +++ Osterosarcoma from 15 yearold female caucasian (IGF-R1 and IGFR-II positive; TPp53 wt, pRb wt,p16-neg; highly aneuploid) H1792 NS ++ ++ Lung Adenocarcinoma(K-RasG12S: TPp53het)) MDA-MD-435 NS ++ +++ Breast carcinoma ofmelanocytic origin (ER-neg, Her2-pos, PR-pos) MG63 NS +++ ++++Osteosarcoma TC32 NS ++ ++ Ewing's sarcoma CHLA9 NS ++ ++ Ewing'ssarcoma CHLA10 NS ++ ++ Ewing's sarcoma TC71 NS ++ ++ Ewing's sarcomaHOS NS +++ ++++ Osteosarcoma PC3 NS ++ ++ Prostate carcinoma SKNMC NS +++++ Ewing's sarcoma MCF-7 NS + ++ Breast carcinoma NS: No staining; +:weak; ++: medium; +++: strong; ++++: Very strong.

TABLE 7 Screening of more human cell cancer cell lines for binding ofrecombinant VAR2CSA (using DBL1-ID2a or ID1-ID2a) Binding was measuredby flow cytometry as described in METHOD 12. Values shown are meanfluorescence intensity using protein concentration of 200 nM. NegativeID1- Cell type control ID2a Comments GP202 21.63 111.37 GastricCarcinoma NCI-N87 7.18 207.72 Gastric Carcinoma MKN45 4.22 55.4 GastricCarcinoma MKN28 6.9 103.84 Gastric Carcinoma AGS 7.25 18.21 GastricCarcinoma KatoIII 7.33 18.76 Gastric Carcinoma SNU-1 4.33 155.79 GastricCarcinoma SNU-638 8.47 8.49 Gastric Carcinoma IPA220 7.72 13.67 GastricCarcinoma MDA-231 3.39 63.43 Triple negative Breast T47D 3.63 48.13Luminal Breast LNCap 6.58 24.86 Prostate PC3 5.2 29.82 Prostate Ovc3161.89 7.24 Ovarian cancer stem cells DBL1- Cell type Blank ID2a NALM-66.19 8.22 Acute lymphatic leukaemia (ALL) 697 3.23 30.36 ALL AMO-1 2.6835.22 Myelomatosis KMM-1 2.82 16.1 Myelomatosis MOLP-8 2.44 19.24Myelomatosis KMS-12-PE 3.02 7.14 Myelomatosis KMS-12-BM 2.2 3.25Myelomatosis U2932 4.24 16.83 Diffuse Large B-cell lymphoma (DLBCL)SU-DHL8 ND 3.75 DLBCL SU-DHL5 2.19 10.28 DLBCL Oci_Ly19 3.38 18.96 DLBCLHBL1 6.53 39.53 DLBCL Farage 2.8 3.28 DLBCL RIVA 2.26 3.32 DLBCLWSU-FSCCL 4.89 22.32 Low-grade follicular small cleaved cell lymphomaU-698-M 2.24 2.85 Lymphoblastic lymphoma del(6)(q15q22)

Example 9 Recombinant VAR2CSA Binds to Cancer Cells with High Affinity

The binding affinity of the recombinant VAR2CSA fragment DBL1-ID2a tothe cancer cell lines, C32 melanoma and two Cho cell lines (described inexample 8) was analysed using a Quartz Crystal Microbalance biosensor(Attana Cell200). A 2-fold dilution series (25-400 nM) of the proteinwas analysed for binding to the cell surface, with regeneration of thebinding surface in between each new protein injection. The bindingaffinity was estimated to lie in the nano-molar range (Table 8), whichis similar to the binding affinity to pure receptor (Table 3).

TABLE 8 Estimated binding affinity (K_(D)) of recombinant DBL1-ID2a (E.coli) to cancer cells expressing CSA (C32 and Cho WT) and lack ofbinding to a CSA-negative cell line (Cho 745) Cell type K_(D) (nM) C32melanoma cells 13 Cho WT 1.4 Cho 745 N/A N/A: K_(D) could not bedetermined due to lack of binding to the cells

Example 10 Recombinant VAR2CSA Protein Binds to Cancer Tissue with HighSpecificity

The binding of recombinant VAR2CSA to primary cancer tissue obtainedfrom human patients is investigated using immunohistochemistry (IHC).The method was developed using human placenta tissue as positive controland Tonsil and liver tissue as negative control. The staining protocolwas optimized on the Ventana Discovery XT platform with no epitoperetrieval. Paraffin embedded tissue spotted on glass slides wasincubated with 0.1-500 nM V5-VAR2CSA (ID1-ID2a) or V5-Control protein(DBL4) for 1 h in room temperature, washed for 8 minutes, incubated with1:700 mouse anti-V5 antibody for 30 minutes, washed for 8 minutes. Boundanti-V5 was subsequently detected using UltraMap anti-mouse HRP.V5-VAR2CSA stains human placenta in 0.5 nM concentrations with nostaining in Tonsil or normal liver. The staining can be completelyblocked by adding 200 μg/μl CSA to the reaction buffer. V5-controlprotein does not stain human placenta tissue at any concentrationstested. A multi-organ tissue micro-array (TMA) representing 24 normalorgans showed low or absent staining when stained with 1 nM V5-VAR2CSA,while cancer specimens of breast, colon, Rectum, Prostate, kidney,liver, bladder, pancreas, squamous cell, Lung, Gall bladder, Stomach,Testis, Ovary, Uterus, Adrenal gland, Thyroid and Thymus, hematopoieticsystem, and the connective tissue (sarcomas) stained positive withintensities equal or higher than human placenta positive control tissue(Table 9).

TABLE 9 Detection of CSA on primary human tumor specimens usingrecombinant VAR2CSA. Table shows number of positive/total number ofcases stained as described in Example 10 for main cancer groups.Positive staining is defined as intensity equal or higher than thatobserved in placenta tissue. Cancer group Positive ratio Bladdercarcinoma 44/56 Prostate carcinoma 71/76 Breast carcinoma 64/75 Melanoma5/6 Sarcoma 23/25 Esophagus Squamous cell carcinoma 2/3 StomachAdenocarcinoma 3/3 Colon carcinoma 2/3 Rectal Adenocarcinoma 3/3 Livercarcinoma 3/3 Renal carcinoma 3/3 Lung carcinoma 2/3 Cervix carcinoma3/3 Ovarian carcinoma 2/3 Diffuse B-cell lymphoma 1/3 Astrocytoma 3/3Pancreatic carcinoma 3/3

Example 11 Inhibition of Transformation-Parameters In Vitro byRecombinant VAR2CSA Proteins

The inhibitory effect of un-coupled VAR2CSA on tumor cell morphology invitro is investigated by three different assays:

-   -   i) The soft agar colony formation assay addresses whether        VAR2CSA can inhibit the ability of cancer cells to proliferate        in a three dimensional matrix.    -   ii) The migration assay addresses whether VAR2CSA can inhibit        the ability of cancer cells to migrate vertically towards a        chemo-attractant in a boyden chamber.    -   iii) The invasion assay addresses whether VAR2CSA can inhibit        the ability of cancer cells to invade through an artificial        basement membrane.

Soft Agar Colony Formation Assay:

Cells are treated with 25-100 nM VAR2CSA for 24 hours before seeded insoft agar matrix, and left for 10-12 days at 37° C. Images are capturedby phase contrast microscope and quantified by ImageJ software.Recombinant VAR2CSA inhibits soft agar colony formation of MG63osteosarcoma and RH30 Rhabdomyosarcoma cells in concentrations between75 and 150 nM.

Basement Membrane Extract (BME)-Coated Cell Invasion Assay:

To model the invasive process, we utilize the CultreCoat® 24 WellBME-Coated Cell Invasion platform (Cedarlane) according to themanufacturer's protocols, with the following modifications. Cells areserum starved one day before assays in the presence or absence of 25-100nM VAR2CSA. On the second day, cells maintained under the aboveconditions are plated in the top chambers (1×10⁵ cells/well) of plates,while lower chambers contained either serum depleted media as a negativecontrol, or media supplemented with 10% FBS. Cells are then incubatedfor another 18 hours. Cells invading through the BME are collected usingdissociation buffer containing Calcein AM, which converts into a highlyfluorescent compound in living cells. Emitted fluorescence are measuredusing a fluorescent plate reader, analyzed by the FLUOStar software,fitted on a standard curve, and converted into corresponding number ofcells.

Migration Assay.

The Migration assay is essentially the same procedure as the BasementMembrane Extract (BME)-coated cell invasion assay, but without BME.

Migration and Invasion capacity of MG63 osteosarcoma, RH30Rhabdomyosarcoma, and MDA-MB-231 triple-negative breast cancer areinhibited by 75-150 nM recombinant VAR2CSA.

Example 12 Analyzing Intracellular Signalling Events Controlling CancerCell Transformation-Parameters Regulated by CSA-Containing Proteoglycans

CSPG4 facilitates proliferation, migration and invasion via a Ras, Rac1and PI3 kinase-dependent mechanism. Based on the results obtained inEXAMPLE 10, we will investigate intracellular signalling events leadingto potential VAR2CSA-mediated inhibition of proliferation, migration andinvasion. This is done with state-of-the-art biochemical and molecularbiology methods including, but not limited to, Rac1 activation assays,immunoblotting of pathway components and in-cell measurements ofreactive oxygen species (ROS) generation. This line of experiments willclarify the signalling pathways affected by VAR2CSA binding toCSA-containing proteoglycans.

Rac1 Activity Assay:

Rac1 activity assays are performed on appropriate human cancer celllines left untreated or treated with recombinant VAR2CSA, according tothe manufacturer's protocols (Thermo Scientific).

Reactive Oxygen Species (ROS) Assays:

Crude ROS levels are measured by CM-H2DCFDA (Invitrogen) according tothe manufacturer's guidelines. Superoxide levels will be measured usingdihydroethidium (DHE). In the presence of the superoxide anion O₂ ⁻,dihydroethidium is rapidly oxidized to oxyethidium, which binds DNA andemits light in the 570-580 nm ranges when excited at 488 nm. For cellculture, after appropriate treatments, cells are washed in Hank'sBalanced Salt Solution (HBSS), incubated for 30-60 minutes in HBSScontaining 10 μM DHE, washed in HBSS and directly analyzed foroxyethidium fluorescence with an epi-fluorescence HAL100 microscope(Zeiss). For tumor sections, snap-frozen tumors are cut in 20 μmsections using a cryostat, washed and DHE-treated as described for celllines, mounted on cover slides and analyzed as for cell lines.Oxyethidium emission are analyzed and quantified using ImageJ software.For all tumor specimens, hematoxylin and eosin (H&E) staining areperformed side-by-side to verify tissue integrity and pathology, usingstandard methods. Preliminary data indicates that recombinant VAR2CSAinhibits ROS-generation in MG63 and U2OS cells.

Immunodetection.

For immunoblotting, proteins separated by SDS-PAGE and transferred to anitrocellulose membrane are detected with relevant primary andappropriate secondary antibodies, ECL Western blotting reagents (ThermoScientific), and film (Kodak and Covance (HA). For microscopy, cells arefixed in 4% formaldehyde, incubated with appropriate primary antibodies,incubated with appropriate secondary FITC-conjugated antibodies andanalyzed by microscopy as described in EXAMPLE 9. Human cancer celllines (MDA-MB-231, MG63, U20S, TC32, TC71 and RH30) were serum starvedfor 24 h with recombinant VAR2CSA (ID1-ID2a) or Control protein (DBL4),and lysates prepared at 0, 1, 2, 3, 4, 5, 6 and 12 h after serum wasadded back to the cells. Using this approach, 100 nM VAR2CSA efficientlyinhibited proto-oncogene tyrosine-protein kinase Src phosphorylation onT416, Focal Adhesion Kinase (FAK) phosphorylation at T397,Extracellular-Signal-regulated Kinase (ERK) 1- and 2-phosphorylation atThr202/Tyr204 for human ERK1 and Thr185/Tyr187 for human ERK2. Thissuggests that recombinant VAR2CSA inhibits canonical ERK signaling incancer cells.

Example 13 Unbiased Analysis of Intracellular Signalling Events Modifiedby Recombinant VAR2CSA

The broad impact of VAR2CSA on intracellular signalling events can beanalysed using expression microarray technology. MG63 osteosarcoma cellswere serum starved for 24 h with no treatment, VAR2CSA or Control (DBL4)and RNA was harvested after 1 h serum addition. The total RNA wasquality tested (RIN<8), used as a template for Affymetrix® probeconstruction and hybridized to the Affymetrix U133Aplus2.0® chip system.This readout provides a snapshot of activated or inactivated signallingpathways after 1 h of serum was added back. Preliminary data confirmedan inhibitory effect on ERK signalling.

Example 14 Inhibition of Cancer Cell Growth In Vivo by RecombinantVAR2CSA Proteins

Based on the results from the in vitro analysis appropriate cell lineswill be selected for in vivo subcutaneous and metastatic xenograftmodels in immuno-compromised mice. The in vivo study addresses five mainquestions:

-   -   i) can i.v. or i.p. administrated recombinant VAR2CSA trace and        bind human cancer cells in vivo?    -   ii) can i.v. or i.p. administration of recombinant VAR2CSA        inhibit tumor formation in vivo?    -   iii) can i.v. or i.p. administration of recombinant VAR2CSA        inhibit growth of established tumors in vivo?    -   iv) can i.v. or i.p. administration of recombinant VAR2CSA        inhibit metastatic spread of human cancer cells in vivo?    -   v) does i.v. or i.p. administration of recombinant VAR2CSA        change CSA-containing proteoglycan-governed signaling events in        human cancer cells in vivo (post mortem pathology and        biochemistry)?

In Vivo Models:

Selected human cancer cell lines representing cancer types showing astrong binding to VAR2CSA are inoculated subcutaneously into Rag2m orSCID immuno-compromised mice at approximately 5×10⁶ cells/animal. Whenthe tumor is established, the mice receive the first injection ofvehicle (Saline) and recombinant VAR2CSA (1 mg/Kg). Treatment isrepeated once a week throughout an experimental period of approximately30 days. Animal weights and tumor volumes are measured every second orthird day and at termination, tumors are removed and divided into twohalves, with one half snap-frozen in liquid nitrogen and the other halffixed in paraffin. Snap-frozen tumors are processed for (DHE) superoxidedetection as described in EXAMPLE 11 (along with correspondinghematoxylin and eosin [H&E] staining of the same tumor specimens).

Example 15 Tracking Micro-Metastasis In Vivo by Tracer-CoupledRecombinant VAR2CSA Peptides

Recombinant VAR2CSA will be coupled to different applicabletracer-molecules in collaboration with external partners or outsourcedon a contract-based agreement. The traceable recombinant VAR2CSAmolecules are analyzed for their ability to track and reportmicro-metastasis in both xenograft and transgenic mouse models. In vivomodels are established as described in EXAMPLE 12. For testing oftracer-coupled VAR2CSA in vivo, mice with metastatic cancer are analyzedby in vivo imaging for the ability of VAR2CSA to track and bindmicro-metastasis.

Example 16 Internalization of Recombinant VAR2CSA Proteins

Recombinant VAR2CSA is internalized by cancer cells. This was shown byfirst conjugating VAR2CSA fragment (DBL1-ID2a) with a fluorophore andthen analysing VAR2CSA uptake both by live imaging and on fixed cells.Cancer cell lines (C32 melanoma and MDA-MB-231) were seeded and grownovernight to 60-80% confluency. Cells were incubated withfluorophore-conjugated VAR2CSA for 10-15 min at 4° C. to allow forsurface binding of VAR2CSA. Cells were then washed to remove unboundVAR2CSA, and subsequently incubated at 37° C. to initiateinternalization for 10 min, 1 h, 2 h, 4 h, and up to 22 h.Fluorphore-conjugated transferrin was used for following classicalclathrin-dependent uptake of transferrin ending up in the lysosomes. Inaddition, for some experiments fluorophore-conjugated dextran was usedfor detecting lysosomes. The live imaging analysis showed that VAR2CSAstarts to reach lysosomes after around 4 h, and after 22 h all VAR2CSAcan be localized to the lysosomal compartments. However, colocalizationof VAR2CSA and transferrin was scarce, and VAR2CSA was taken up muchslower than transferrin. The fact that recombinant VAR2CSA is taken upby cancer cells, allows us to fuse or conjugate VAR2CSA to cytotoxiccompounds that become active inside the cancer cell. Table 10 summarizesthe result from indicated cancer cell lines tested for internalizationof recombinant VAR2CSA.

TABLE 10 Cells were incubated with medium alone (blank) or recombinantproteins (DBL1-ID2a or ID1-ID2a) at 75 nM for 1 h, followed byincubation with anti-V5-FITC (Invitrogen) at 1:800, cells were washedthrice between each incubation. Shown are the medium score of FITCfluorescence intensity at either the plasma membrane or intracellularstructures recorded from a minimum of 4 high power field images using aHAL100 Zeiss microscope. Plasma membrane Intracellular localizationlocalization Cell line (after 1 h) (after 1 h) U2OS + ++++ RH30 + +++MG63 + ++++ MeWo + ++++ HOS + +++ MDA-MB-231 + ++++ SKNMC ++++ (+)RH18 + +++ TC71 + ++ TC3 + ++ Scoring system is: +: weak;, ++: medium;+++: strong; ++++: Very strong.

Example 17 Fused VAR2CSA-Toxin Protein Kills Cancer Cells

DBL1-ID2a and ID1-ID2a VAR2CSA gene fragments have been fused toPseudomonas exotoxin A and diptheria toxin as various constructs (SEQ IDNO:60-70, 72). These fused VAR2CSA-toxin proteins are expressed in E.coli. The protein construct called BPTI-ID1-ID2aFCR3-PE38LR (SEQ IDNO:60), which is based on ID1-ID2a from VAR2CSA and PE38 has beensuccessfully produced and analysed for binding to cancer cells (Table11) as well as cytotoxic activity as described in Method 13.

Preliminary data show that this fused VAR2CSA-toxin protein binds toCSA-expressing cancer cells and is able to induce cell death (IC50 forthe U2OS cell line is below 1 nM.

TABLE 11 Binding of VAR2CSA-PE38 to cancer cells analysed by flowcytometry Binding of DBL1-ID2a (naked protein) and ID1-ID2a-PE38 at 200nM to Myla2059 cells (T cell lymphoma) was detected with anti-PENTA HISantibody and anti-mouse-FITC antibody and analysed by flow cytometry.Binding is given as mean fluorescence intensity (MFI). DBL1-ID2aID1-ID2a-PE38 Control^(c) Binding to cells 24.7 12.4 2.3 Binding totreated cells^(a) 4.4 2.5 2.5 Inhibition of binding^(b) 3.2 2.1 —^(a)cells were treated with chondroitinase ABC to remove CS chains fromcell surface, ^(b)protein was mixed with soluble CSA (400 ug/m) prior toaddition to cells, ^(c)control equals cells stained with the first andsecond layer of antibodies only.

Example 18 Analyzing the Anti-Tumor Effect of Cytotoxic CompoundsCoupled to Recombinant VAR2CSA

Based on the results in EXAMPLE 14 recombinant VAR2CSA will be soughtcoupled to relevant cytotoxic compounds and tested in vivo forperformance. Coupling of relevant compounds to VAR2CSA will be performedin collaboration with external partners or outsourced on acontract-based agreement. In particular we analyze whether theseVAR2CSA:compound-fusions can:

-   -   i) be delivered specifically to the tumor environment in vivo.    -   ii) be up-concentrated and retained specifically in the tumor        environment in vivo.    -   iii) specifically kill tumor cells with minimal damage to normal        tissues in vivo.

In vivo models are established as described in EXAMPLE 12. The mice aretreated with cytotoxic VAR2CSA conjugates, and the effect is assayed asdescribed for un-conjugated protein in EXAMPLE 12.

Example 19 Purification of CSA-Expressing Stem Cells from HeterogeneousCell Populations

Pluripotent stem cells have been reported to express high levels ofCSPG4. Stem cells also express other CSA-containing proteoglycans, suchas CD44, which VAR2CSA can bind to. Accordingly, recombinant VAR2CSAwill be conjugated to an appropriate resin (beads), mixed with aheterogeneous but stem cell or cancer stem cell-containing cellpopulation and sought purified by conventional centrifugation protocols.Purified cells will be analyzed for expression of diverse stem cellmarkers including CD44, CD31, CD4, OCT4, SOX2, Nestin and Nanog, byimmunoblotting (as in EXAMPLE 11), microscopy and FACS (as in EXAMPLE9). A common trait of cancer stem cells is high expression of Aldehydedehydrogenase 1A (ALDH1 High). This can be conveniently measured usingthe AldeFluor® Kit (Stem Cell Technologies). Recombinant VAR2CSA bindingto MDA-MB-231 detects a subpopulation of ALDH1 High cells, suggestingthat VAR2CSA can bind human cancer stem cells.

Example 20 Identification and Targeting of CD44-Expressing Cancer StemCells

CD44 is currently the most popular marker for cancer stem cells and itis a CSA-containing proteoglycan that can bind recombinant VAR2CSA. Byusing the same approaches as in EXAMPLE 12-15, it will be investigatedwhether un-modified and modified recombinant VAR2CSA peptides canlocate, bind, purify and potentially kill the highly resistantCD44-positive cancer stem cells.

Example 21 Detection of Circulating Tumor Cells

We will examine whether recombinant VAR2CSA can be used as a prognosticmarker for cancer recurrences. Cancer cells spread through the bloodsystem after detachment from the primary tumor. A subsequent risk of theoccurrence of circulating tumor cells (CTCs) is extravasation andmetastasis. Current assays used for detecting CTCs have a poorsensitivity and cannot be directly correlated with risk of metastases.Using VAR2CSA-coupled magnetic beads and flow cytometry, we willinvestigate the prognostic value of detecting CS expressing cancer cellsin the blood flow. This method could be used as a fast and painlessexamination of patients.

Example 22 Identification of Potential CSPG Molecules that were Targetedby VAR2CSA

Recombinant VAR2CSA protein (DBL1-ID2a) with a V5-tag was screened forbinding to a panel of transfected HEK293 cells expressing >3000 humanmembrane receptors. A set of 25 receptors have been identified aspotential targets of VAR2CSA (Table 12). The interaction between VAR2CSAand these receptors will be further verified by analysis of the bindingspecificity through inhibition with CSA and HS, both in the HEK293system and in ELISA.

TABLE 12 Receptors that were experimentally identified as potentialtargets of VAR2CSA. Gene ID Name UniProt/SwissProt BCAN Brevican PGCBHUMAN, Q96GW7 BDKRB2 Bradykinin receptor B2 BKRB2 HUMAN, P30411 CA9Carbonic anhydrase IX CAH9 HUMAN, Q16790 CCR10 chemokine (C-C motif)receptor 10 CCR10 HUMAN, P46092 CD44 CD44 molecule (Indian blood group)CD44 HUMAN, P16070 CDH8 Cadherin 8, type 2 CADH8 HUMAN, P55286 CFBComplement factor B CFAB HUMAN, P00751 GABBR2 gamma-aminobutyric acid(GABA) B GABR2 HUMAN, O75899 receptor, 2 GPC3 Glypican 3 GPC3 HUMAN,P51654 GPC5 Glypican 5 GPC5 HUMAN, P78333 GPR65 G-protein coupledreceptor 65 PSYR HUMAN, Q8IYL9 GPRC5B G protein-coupled receptor, familyGPC5B HUMAN, Q9NZH0 C, group 5, member B KCNA2 potassium voltage-gatedchannel, KCNA2 HUMAN, P16389 shaker-related subfamily, member 2 PKD2polycystic kidney disease 2 PKD2 HUMAN, Q13563 (autosomal dominant)PODXL2 podocalyxin-like 2 PDXL2 HUMAN, Q9NZ53 PTPRG protein tyrosinephosphatase, PTPRG HUMAN, P23470 receptor type, G S100A9 S100 calciumbinding protein A9 S10A9 HUMAN, P06702 SDC1 Syndecan 1 SDC1 HUMAN,P18827 SDC4 Syndecan 4 SDC4 HUMAN, P31431 STX2 Syntaxin 2 STX2 HUMAN,P32856 STXBP5 syntaxin binding protein 5 STXB5 HUMAN, Q5T5C0 (tomosyn)TGFBR3 transforming growth factor, beta TGBR3 HUMAN, Q03167 receptor IIITMEFF1 transmembrane protein with EGF- TEFF1 HUMAN, Q8IYR6 like and twofollistatin-like domains 1 TMEFF2/TENB2 transmembrane protein with EGF-TEFF2 HUMAN, Q9UIK5 like and two follistatin-like domains 2 TMEM154Transmembrane protein 154 (None) BCAN Brevican PGCB HUMAN, Q96GW7 BDKRB2Bradykinin receptor B2 BKRB2 HUMAN, P30411 CA9 Carbonic anhydrase IXCAH9 HUMAN, Q16790 CCR10 chemokine (C-C motif) receptor 10 CCR10 HUMAN,P46092 CD44 CD44 molecule (Indian blood group) CD44 HUMAN, P16070 CDH8Cadherin 8, type 2 CADH8 HUMAN, P55286 CFB Complement factor B CFABHUMAN, P00751 GABBR2 gamma-aminobutyric acid (GABA) B receptor, 2 GABR2HUMAN, O75899 GPC3 Glypican 3 GPC3 HUMAN, P51654 GPC5 Glypican 5 GPC5HUMAN, P78333 GPR65 G-protein coupled receptor 65 PSYR HUMAN, Q8IYL9GPRC5B G protein-coupled receptor, family C, group 5, GPC5B HUMAN,Q9NZH0 member B KCNA2 potassium voltage-gated channel, shaker-relatedKCNA2 HUMAN, P16389 subfamily, member 2 PKD2 polycystic kidney disease 2(autosomal PKD2 HUMAN, Q13563 dominant) PODXL2 podocalyxin-like 2 PDXL2HUMAN, Q9NZ53 PTPRG protein tyrosine phosphatase, receptor type, G PTPRGHUMAN, P23470 S100A9 S100 calcium binding protein A9 S10A9 HUMAN, P06702SDC1 Syndecan 1 SDC1 HUMAN, P18827 SDC4 Syndecan 4 SDC4 HUMAN, P31431STX2 Syntaxin 2 STX2 HUMAN, P32856 STXBP5 syntaxin binding protein 5(tomosyn) STXB5 HUMAN, Q5T5C0 TGFBR3 transforming growth factor, betareceptor III TGBR3 HUMAN, Q03167 TMEFF1 transmembrane protein withEGF-like and two TEFF1 HUMAN, Q8IYR6 follistatin-like domains 1TMEFF2/TENB2 transmembrane protein with EGF-like and two TEFF2 HUMAN,Q9UIK5 follistatin-like domains 2 TMEM154 Transmembrane protein 154(None) THBD Thrombomodulin TRBM HUMAN, P07204 CSPG5 chondroitin sulfateproteoglycan 5 CSPG5 HUMAN, O95196 (neuroglycan C) STXBP5 syntaxinbinding protein 5 (tomosyn) STXB5 HUMAN, Q5T5C0

DISCUSSION

Malaria is one of the most common infectious diseases and one of thelargest global health problems. Pregnant women are especially vulnerableto infection, despite previously acquired immunity. In this study wehave addressed key questions related to the molecular mechanism behindthe VAR2CSA-CSA interaction in PM.

Previous work has suggested that the minimal CSA binding region inVAR2CSA is DBL2X-ID2b, with the need for DBL1X or DBL3X for fullaffinity binding (Dahlback, M., Jorgensen, L. M., Nielsen, M. A.,Clausen, T. M., Ditlev, S. B., Resende, M., Pinto, V. V., Arnot, D. E.,Theander, T. G., and Salanti, A. J Biol Chem 286, 15908-15917). Incontinuation of this work we made further truncations of VAR2CSA,focusing on the DBL2X region. We show that the core CSA-binding sitelies within the DBL2X domain including small parts of the flankinginterdomain regions. The binding does not depend on the ID2b region, oron the DBL1X or DBL3X flanking domains, as previously suggested. This isevident by the specific CSPG binding of ID1-ID2a and ID1-DBL2Xb (Table3). The minimal binding region is ID1-DBL2Xb, which bound CSPG withcharacteristics comparable to that of full-length VAR2CSA.

It is interesting that these new data maps the core-CSA binding siteonto a single domain. Binding of DBL2X (and any other single DBL domain)to CSA has previously been shown to be non-specific and of weak affinity(Resende, M., Ditlev, S. B., Nielsen, M. A., Bodevin, S., Bruun, S.,Pinto, V. V., Clausen, H., Turner, L., Theander, T. G., Salanti, A., andDahlback, M. (2009) Int J Parasitol 39, 1195-1204). It is clear that theID1 and parts of the ID2a interdomains are essential for CSA binding.DBL1X-DBL2Xa and ID1-DBL2Xa did not bind CSPG. The two C-terminal DBL2Xborders (DBL2Xa and DBL2Xb) differ by 93 amino acids. Since deletion ofthese amino acids eliminates binding they must be important for CSAbinding.

The ID1-DBL2Xb minimal binding region is much smaller than full-lengthVAR2CSA, having a molecular weight of only 62 kDa. It is unlikely thatfurther substantial truncations of VAR2CSA will be functional in bindingCSA. Our data redefines DBL2X as a larger functional domain,incorporating parts of the flanking ID1 and ID2a interdomains.

A VAR2CSA based vaccine against PM must be able to induce a strongprotective immune response. In this, the most important aspect is thegeneration of IgG antibodies capable of inhibiting placental-specificparasite adhesion. To test the immunogenic characteristics of ourproduced fragments, we used them in the immunization of rats. Seraraised against all fragments containing the CSA binding site inhibitedparasite adhesion to CSA. Importantly, sera raised against ID1-ID2aresulted in almost complete inhibition. This suggests that the minimalCSA binding fragments retain the capacity for inducing a stronganti-adhesive immune response. This conclusion was further supported bythe fact that antibodies purified from anti-FV2 serum on ID1-ID2aretained most of the adhesion blocking activity, and that theanti-ID1-ID2a antibody depleted anti-FV2 sample lost most of itsactivity. This indicates that epitopes required for the induction ofadhesion blocking antibodies are located within this region.

In this study we have tested anti-VAR2CSA sera in homologous inhibitionof FCR3 parasites binding to CSA. It is important that a vaccine iscapable of inhibiting placental adhesion regardless of parasite strainorigin. A major concern in vaccine development is therefore the highinterclonal diversity among parasite variants. While recombinantfull-length VAR2CSA is very immunogenic the antibodies produced are notcross-inhibitory (Avril, M., Hathaway, M. J., Srivastava, A.,Dechavanne, S., Hommel, M., Beeson, J. G., Smith, J. D., and Gamain, B.PLoS One 6, e16622). A recent study shows that DNA-vaccination withID1-DBL2X from FCR3, induces antibodies that are cross-inhibitory,inhibiting CSA adhesion of other laboratory strains as well as parasitesisolated in the field (Bordbar, B., Tuikue-Ndam, N., Bigey, P.,Doritchamou, J., Scherman, D., and Deloron, P. Vaccine). This supportsthe use of this small fragment in a PM vaccine.

Cardin and Weintraub predicted that a GAG binding site would take one oftwo forms (Cardin, A. D., and Weintraub, H. J. (1989) Arteriosclerosis9, 21-32). These are X-B-B-X-B-X and X-B-B-B-X-X-B-X, where X is anyhydropathic residue and B is any basic residue, with a preference forarginine. Both of these describe a binding site for a sulfateddisaccharide. While many interactions may occur, the ionic interactionbetween negatively charged sulfates and basic amino acids are thought tobe most important. We mutated two such sites within the minimal bindingregion; 625-GKNLKKRY-632 in DBL2X and 458-NKKKECKD-465 in ID1. We alsodeleted a large region within a dimorphic sequence motif (DSM) locatedin the N-terminal part of DBL2X, as this has been suggested to have afunction in binding. Deletion of the DSM region had no affect on CSAbinding. Neither did any substitutions in the putative GAG bindingsites. This is a clear indication that these sites have little or nofunction in CSA binding.

It has been shown that the minimal binding requirement for the human CSAreceptor is a dodecasaccharide with 2-4 C4 sulfated GaINAcmonosaccharides (Alkhalil, A., Achur, R. N., Valiyaveettil, M.,Ockenhouse, C. F., and Gowda, D. C. (2000) J Biol Chem 275,40357-40364). It is remarkable that the VAR2CSA expressing parasites, invivo, are very specific for CSA carrying only 2-8% C4 sulfateddisaccharide units. To examine if the VAR2CSA-CSA complex formation isdependent on ionic interactions, we tested binding at different saltconcentrations. Binding of ID1-ID2a, DBL1X-ID2a and FV2 in 150 mM-300 mMNaCl show a linear relationship when plotting Log (K_(D,observed)) vs.Log [Na⁺]. We find that binding depends on a maximum of 2-3 ionicinteractions. It is interesting that the value for the full-lengthprotein is higher than for the shorter fragments, indicating that thisprotein makes an additional ionic interaction with CSA. We have in thisstudy screened for fragments containing the CSA specific high-affinitybinding region. It is possible that more interactions occur indownstream regions of the protein, but that the core site lies withinDBL2X. Extrapolating and finding the Y intercept ([Na+]=1 M, Log[Na⁺]=0) tells us that K_(D,nonionic)=5.9 μM for FV2, K_(D,nonionic)=3.4μM for DBL1X-ID2a, and K_(D,nonionic)=0.7 μM for ID1-ID2a. Thisindicates that only 25-30% of the VAR2CSA-CSA binding can be accountedfor by ionic interactions. This is in contrast to other GAG bindingproteins, which have shown up to 80-90% dependency on ionic interactionsin similar assays (Faller, B., Mely, Y., Gerard, D., and Bieth, J. G.(1992) Biochemistry 31, 8285-8290; Hileman, R. E., Fromm, J. R., Weiler,J. M., and Linhardt, R. J. (1998) Bioessays 20, 156-167).

Our data suggest that the VAR2CSA-CSA interaction does not conform toconventional GAG-protein interactions. We hypothesize that the high CSAaffinity is achieved through a multivalent interaction, which mayinclude multiple binding sites making nonionic interactions with the CSAcarbohydrate backbone. Some of the interaction is ionic and some degreeof sulfation is needed for VAR2CSA binding. It is therefore likely thatthere is an interaction between basic amino acids and sulfates, but thatthis is not the determining factor in the affinity.

In this study we have defined a small single-domain VAR2CSA fragmentthat can be produced in eukaryotic cells as a functional CSA-bindingprotein, and has the capacity to induce highly adhesion-blockingantibodies. This fragment has the potential to be a powerful candidatefor a vaccine against PM.

The data identifies a small recombinant part of VAR2CSA that bindsspecifically to CSA thereby mediating placental binding of infectederythrocytes. We show that this VAR2CSA fragment also binds specificallyto cancer cells, through an interaction with CSA presented on CSPG4 orother protein backbones that were identified in this study. In addition,we find that binding of VAR2CSA polypeptides, based on this smallfragment, to cancer cells inhibits migration and invasion of the cells.These VAR2CSA polypeptides also inhibit canonical ERK signaling, and wefind that VAR2CSA polypeptides that are fused to a toxin efficientlykill the cancer cells.

Methods

Method 1—Cloning and Protein Expression in Insect Cells

VAR2CSA sequence fragments were amplified from codon optimized FCR3(GenBank accession no. GU249598) or 3D7 (GenBank accession no. JQ247428)VAR2CSA genes using specific primers (Table 2). Simple fragments wereamplified in a one-step PCR. Amino acid substitution constructs weremade in a two-step PCR. First PCR amplified two fragments from the codonoptimized FCR3 template, containing overlapping complimentary ends.Second PCR amplified the total construct, using the two overlappingfragments as template with primers specific for the outer borders. Allfragments were sequenced for verification. Fragments were cloned intothe baculovirus vector pAcGP67-A (BD Biosciences), modified to contain aV5 and His tag at the C-terminal. The proteins were expressed inbaculovirus-infected insect cells as soluble protein secreted into thecell culture supernatant. Briefly, linearized Bakpak6 Baculovirus DNA(BD Biosciences) was co-transfected with the pAcGP67-A plasmids, intoSf9 insect cells for generation of recombinant virus particles. 10 ml ofthe second amplification was used to infect High-Five cells in 400 mlserum-free medium (10486, GIBCO) at a density of 1×10⁶ cells/ml. Thesecreted recombinant protein was harvested from the supernatant 3 daysafter initial infection. The supernatant was filtered (0.2 μm), dialyzedand concentrated before protein purification.

Method 2—Protein Purification and SDS-PAGE

The filtered supernatant containing the secreted recombinant protein wasdialyzed using an ÄKTA cross-flow (GE Healthcare). The dialysis wasperformed in 10 mM NaH₂PO₄ (pH 7.4, Sigma-Aldrich) and 500 mM NaCl. Theresulting solution was filtered (0.2 μm) and imidiazole was added to afinal concentration of 15 mM. The protein was then purified on a 1-mlHisSelect column (H8286, Sigma-Aldrich). Bound protein was eluted with10 mM NaH₂PO₄(pH 7.4), 500 mM NaCl, and 500 mM imidiazole. Proteinsneeded for Quartz Crystal Microbalance measurements and SAXS werefurther purified to obtain monomers by size exclusion chromatographyusing a HiLoad 16/60 Superdex 200 column (GE Healthcare) in 20 mM Tris(pH 8) and 200 mM NaCl. The purity and structural integrity of theprotein was verified by SDS-PAGE.

Method 3—ELISA

Falcon microtiter plates (351172, BD Biosciences) were incubated at aconcentration of 3 μg/ml for CSPG (bovine) (D8428, Sigma) or HSPG(H4777, Sigma) and 100 μg/ml for CSA (C9819, Sigma), CSC (400675,Seikagaku), and CSB (C3788, Sigma) overnight at 4° C. The plates werethen blocked with TSM binding buffer (20 mM Tris, 150 mM NaCl, 2 mMCaCl₂, 0.05% Tween-20, 1% BSA, PH7.4 at 25° C.) for 2 hours at 37° C. ona shaker. A 2-fold dilution series (1.56 mM-100 mM) of protein wasprepared in TSM binding buffer and added to the plates, which wasincubated 1 hr at 37° C. on a shaker. All measurements were performed intriplicates. The plates were washed three times in TSM washing buffer(20 mM Tris, 150 mM NaCl, 2 mM CaCl₂, 0.05% Tween-20, PH7.4 at 25° C.).The plates were then incubated with 1:3000 anti-V5-HRP antibody (R96125,Invitrogen) in TSM binding buffer 1 hr at 37° C. on a shaker. The plateswere washed three times in TSM washing buffer. Finally the plates weredeveloped with o-phenylenediamine substrate (DAKO) for 15 min. Thereaction was quenched with 2.5M H₂SO₄. Absorbance was measured at 490nm.

Method 4—Quartz Crystal Microbalance (Attana A100)

Experiments were performed on an Attana A100 (Attana AB), using goldplated 10 MHz, AT-cut quartz crystal, polystyrene chips (3611-3103Attana AB). All buffers and reagents were filtered to 0.2 μm. The ligandwas CSPG (Bovine) (D8428, Sigma) or HSPG (H4777, Sigma), coated at aconcentration of 100 μg/ml. Coating was done in steady state by addingligand solution and incubation 30 minutes at room temperature. This wasfollowed by blocking the plate with PBS containing 0.1% Ig-free BSA(BSA-50, Rockland), 30 minutes at room temperature. The Attana A100 waswashed with 1% SDS prior to every experiment, using the manufacturerspredefined daily wash program. Following the wash, the running bufferwas switched to PBS at a flow rate of 254/min, at 25° C., and themachine was allowed to stabilize at a maximum change in frequency of 0.5Hz/min. Once stabilized PBS was injected multiple times to show that theinjection process minimally affected the baseline. Prior to sampleinjection PBS was injected as a blank. Analyte was injected in a 1:3dilution series (0.25 μg/ml-60 μg/ml) starting with the lowestconcentration. Association time was set to 84 seconds and disassociationtime to 5 minutes. Due to high affinity of binding it was not possibleto regenerate binding surface following injections. The data collectedwas processed in the Attester Evaluation software (Attana AB). Curveswere fitted in a simple 1:1 model. k_(on) and k_(off) were determined bycurve fitting and K_(D) was calculated based K_(D)=K_(off)/K_(on).

Method 5—Salt Titration Assay

The ionic dependency of VAR2CSA-CSA binding was tested in an ELISA basedbinding assay. CSPG was coated at 3 μg/ml. A 1:2 dilution series(400-1.56 nM) of protein was added in several different NaClconcentrations (150 mM, 200 mM, 250 mM, and 300 mM). All experimentswere performed in triplicates. The K_(D) values were calculated for eachtitration series in Graphpad Prism using non-linear regression (Leastsquares fit with hill slope).

Method 6—Animal Immunizations and Serum Extraction

All animal immunizations complied with national and Europeanregulations. Wistar rats were injected subcutaneously with 30 μgrecombinant protein in Freunds complete adjuvant (F5881, Sigma-Aldrich).The immunization was boosted three times at 3-week intervals with 15 μgprotein in Freunds incomplete adjuvant (F5506, Sigma-Aldrich). Bloodsamples were taken one week after each boost, and serum was extracted bycentrifugation.

Method 7—IgG Affinity Purification

Pools of sera from rats immunized with full-length FCR3 VAR2CSA (FV2)were affinity purified on 1 ml NHS-activated HP column (HiTrapNHS-activated HP, 17-0716-01, GE Healthcare), containing immobilizedmultidomain FCR3 proteins (DBL1X-DBL2Xa, DBL1X-ID2a, ID1-ID2a, orID1-DBL4s) and full-length FV2. Purification was done according to themanufacturer's protocol. In short, coupling of ligand to column was doneby adding 1 ml 1:1 solution of coupling buffer (0.2 M NaHCO₃, 0.5 MNaCl, pH 8.3) and ligand (concentration 0.5-10 mg/ml) to the column. Thecolumn was sealed and incubated for 30 min at room temperature, followedby incubation at 4° C. overnight. The column was washed with 6 ml BufferA (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3), 6 ml Buffer B (0.1 Macetate, 0.5 M NaCl, pH 4) and finally 6 ml Buffer A. After anincubation period of 30 min at room temperature, the washing wasrepeated in reverse order (Buffer B, A, B). 8-10 ml PBS was injected toadjust the pH before purifying the serum. The sample was passed throughthe column 3-5 times. The column was washed with 10 ml PBS beforeantibodies were eluted with 10 ml elution buffer (0.1 M citric acid, pH2.7).

Method 8—P. falciparum Parasite Cultures

P. falciparum FCR3 type parasites were maintained in culture using 5%hematocrit (Human Blood-group 0 Rh+) in parasite medium RPMI-1640(BE12115F, Lonza) supplemented with 25 mM NaHCO3, 0.125 μg/ml gentamycinsulfate (BE02012E, Lonza), 0.125 μg/ml AIbuMAX II (Ser. No. 11/021,029,Invitrogen) and 2% normal human serum. IEs were repeatedly panned onBeWo cells (CCL98, ATCC) to maintain the CSA adhering phenotype.Furthermore, isolates were tested to be mycoplasma negative and wereregularly genotyped by PCR using nested GLURP (Glutamate-rich protein)and MSP-2 (Merozoite surface protein 2) primers.

Method 9—Purification of Late Stage Trophozoites

Parasite cultures were enriched for late trophozoite and schizont stagein a strong magnetic field using a MACS CS-column (130-041-305, MiltenyiBiotec) and a Vario-MACS magnet (Miltenyi Biotec). In brief, theparasite culture suspension was applied to the column. The column wasthen washed with 2% fetal calf serum (F6178, Sigma-Aldrich) in PBS.Late-stage infected erythrocytes were elute from the column afterseparation from the magnet, spun down and resuspended in 2% fetal calfserum in PBS and diluted to a concentration of 2×10⁶ IEs/ml.

Method 10—Flow Cytometry (FCM)

Antibody binding to native VAR2CSA on the purified late-stagetrophozoites infected erythrocytes, was measured by flow cytometry(FCM). 100 μl purified late-stage parasites at a concentration of 2×10⁵IEs/ml in PBS with 2% FCS were labeled with serum (depleted fornon-specific binding by pre-incubation with non-infected erythrocytes)in a final concentration of 1:10. The cells were washed three times inPBS with 2% FCS. The cells were then further labeled with ethidiumbromide (Ser. No. 15/585,011, Invitrogen) in a final concentration of 2μg/ml and a 1:100 dilution of FITC labeled secondary anti-rat-IgGantibody (62-9511, Invitrogen). As negative controls, late-stageparasites were also incubated with serum from rats immunized with anantigen other than VAR2CSA and with secondary antibodies alone. Datafrom 5000 ethidium bromide positive IEs were collected using a FC500flow cytometer (Beckmann Coulter). Finally the median fluorescenceintensity was determined using the WinList 5.0 software (Verify SoftwareHouse).

Method 11—Inhibition of Parasites Binding CSPG

Serum antibodies were analyzed for their ability to inhibit IE bindingto CSPG. This was done in a 96-well plate format using arobot-standardized washing method. Wells were coated with 2 μg/ml CSPG(D8428, Sigma-Aldrich). A total of 2×10⁵ tritium labeled (HypoxanthineMonohydrochloride, PerkinElmer, NET177005MC) late□stage IEs in 100 μLwere added in triplicates to the wells. The labeled IEs were thenincubated with serum for 90 min at 37° C. Unbound IEs were washed awayby a pipetting robot (Beckman Coulter). The proportion of adhering IEswas determined by liquid scintillation counting on a Topcount NXT(Perkin□ Elmer).

Method 12a—Cancer Cell Binding Assays

Flow-cytometry (FCM) was used to test the reactivity of the VAR2CSAminimal binding polypeptide to CSPG expressed on the surface of variouscell lines. Cells were cultured in RPMI supplemented with 10% foetalcalf serum (CHO cells, C32), Hams F12 (BeWo), kept in 5% carbon dioxideat 37° C. or purified from a human blood sample in CPD buffer (red bloodcells). Aliquots of cells (1×10⁵) were sequentially exposed to theVAR2CSA minimal binding polypeptide (150, 75 or 37 nM) and α-V5-FITC(1:800)(Invitrogen) diluted in FACS2 (PBS+2% FCS) for 30 minutes at +4 Cin dark with smooth agitation. As negative controls a truncated versionof the minimal binding polypeptide and FACS2 buffer were used. Intactcells were gated based on the forward and side scatter signal. Data wereacquired using a FC500 flow-cytometer (Beckman Coulter) from a minimumof 5000 cells. All samples relating to a particular cell line wereprocessed and analyzed in a single assay.

Method 12b—Cancer Cell Binding Assays

As an alternative to the flow-cytometry assay above, cells wereincubated with VAR2CSA minimal binding polypeptide and α-V5-FITC(1:500)(Invitrogen) diluted in HBSS. VAR2CSA polypeptide was used at thesame concentrations as written above. Following α-V5-FITC staining cellswere washed 3 times in HBSS, collected in Enzyme-free cell detachmentbuffer (Invitrogen) and analyzed on a FACS Calibur (BD Biosciences) forFL-1 signal intensity.

Abbreviations CIDR, cysteine-rich inter-domain region; CSA, chondroitinsulfate A; CSPG, chondroitin sulfate proteoglycan; DBL, Duffybinding-like domain; FCM, flow-cytometry; FV2, full-length ecto-domainof the VAR2CSA protein without N-terminal segment; HSPG, heparan sulfateproteoglycan; ID, inter-domain; IE, P. falciparum-infected erythrocyte;NTS, N-terminal segment; PM, placental malaria; PfEMP1, Plasmodiumfalciparum erythrocyte membrane protein 1; PM, placental malaria.

Method 13—Cytotoxicity Test In Vitro of Fused VAR2CSA-Toxin Proteins

Cancer cell lines were seeded in a 96-well plate, with 500.0 cells/wellone day before the experiment. On the day of experiment a 10-folddilution series (ranging from 10 μg/ml to 0.01 ng/ml) of fusedVAR2CSA-toxin, and control protein (VAR2CSA without toxin) was added toseparate wells. Similar dilution series, which also contained 400 μg/mlof CSA, was made for both proteins and added to separate wells. Thecells with proteins were incubated for 72 hours at 37° C. Cell death wasanalyzed by a MTT cell proliferation assay, where readout is absorbanceat 570 nm.

Method 14—Staining of Paraffin-Embedded Human Tissue Samples

The binding of recombinant VAR2CSA to primary cancer tissue obtainedfrom human patients is investigated using immunohistochemistry (IHC).Paraffin embedded tissue spotted on glass slides subjected no antigenretrieval was incubated with 0.1-500 nM V5-VAR2CSA variants orV5-Control protein (DBL4) for 1 h in room temperature, washed for 8minutes, incubated with 1:700 mouse anti-V5 antibody for 30 minutes,washed for 8 minutes. Bound anti-V5 was subsequently detected usingUltraMap anti-mouse HRP using the Ventana Discovery XT platform.

1. A method for the treatment of any indication associated withexpression, such as inappropriate expression of CSA, such as in cancer,arthritis, arthrosis, multiple sclerosis, pathological conditions causedby neural damage, conditions of the cartilage and scar tissue, such asin rheumatism, cartilage repair or wound healing, or in psoriasis; themethod comprising administering a therapeutically or prophylacticallyeffective amount of a VAR2CSA polypeptide, or a conjugate or fusionprotein comprising a VAR2CSA polypeptide and a therapeutic or diagnosticeffector moiety, such as a cytotoxic moiety, fluorescent label, and/or aradiolabel; to a subject in need thereof.
 2. A method for preventing theoccurrence of an indication or condition associated with expression,such as inappropriate expression of CSA, such as in cancer, multiplesclerosis, arthritis, arthrosis, pathological conditions caused byneural damage, conditions of the cartilage and scar tissue, such as inrheumatism, cartilage repair or wound healing, or in psoriasis; themethod comprising administering a therapeutically or prophylacticallyeffective amount of a VAR2CSA polypeptide, or a conjugate or fusionprotein comprising a VAR2CSA polypeptide and a therapeutic or diagnosticeffector moiety, such as a cytotoxic moiety, fluorescent label, and/or aradiolabel; to a subject in need thereof.
 3. The method of claim 1,wherein said VAR2CSA polypeptide is a fragment of VAR2CSA, whichfragment consist of a sequential amino acid sequence of ID1, and DBL2Xb,and optionally ID2a.
 4. The method according to claim 3, which fragmentcomprises an amino acid sequence having at least 70% sequence identitywith any one amino acid sequence of 1-577 of SEQ ID NO:1, 1-592 of SEQID NO:3, 1-579 of SEQ ID NO:4, 1-576 of SEQ ID NO:5, 1-586 of SEQ IDNO:10, 1-579 of SEQ ID NO:11, 1-565 of SEQ ID NO:29, 1-584 of SEQ IDNO:34, 1-569 of SEQ ID NO:36, 1-575 of SEQ ID NO:37, 1-592 of SEQ IDNO:38, 1-603 of SEQ ID NO:41, 1-588 of SEQ ID NO:43, 1-565 of SEQ IDNO:44, 1-589 of SEQ ID NO:45, 1-573 of SEQ ID NO:48, 1-583 of SEQ IDNO:53, or 1-569 of SEQ ID NO:54.
 5. The method according to claim 3,which fragment comprises an amino acid sequence having at least 70%sequence identity with an amino acid sequence of 578-640 of SEQ ID NO:1,593-656 of SEQ ID NO:3, 580-643 of SEQ ID NO:4, 577-640 of SEQ ID NO:5,587-650 of SEQ ID NO:10, 580-643 of SEQ ID NO:11, 566-628 of SEQ IDNO:29, 585-647 of SEQ ID NO:34, 570-632 of SEQ ID NO:36, 576-639 of SEQID NO:37, 593-655 of SEQ ID NO:38, 604-667 of SEQ ID NO:41, 589-652 ofSEQ ID NO:43, 566-628 of SEQ ID NO:44, 590-653 of SEQ ID NO:45, 574-637of SEQ ID NO:48, 584-646 of SEQ ID NO:53, or 570-632 of SEQ ID NO:54. 6.The method according to claim 1, wherein the cytotoxic moiety isselected from calicheamycin, auristatin, doxorubicin, maytansinoid,taxol, ecteinascidin, geldanamycin, methotrexate and their derivatives,cytotoxic proteins such as Pseudomonas exotoxin A, diphtheria toxin,ricin toxin, pokeweed antiviral protein, saporin, gelonin and functionalvariants, fragments, and combinations thereof.
 7. The method accordingto claim 1, wherein the therapeutic or diagnostic effector moiety is ananti-inflammatory agent.
 8. The method according to claim 1, wherein thetherapeutic or diagnostic effector moiety is CSPG4, CD44, or otherproteoglycans exemplified but not limited to those in Table
 1. 9. Themethod of claim 2, wherein said VAR2CSA polypeptide is a fragment ofVAR2CSA, which fragment consist of a sequential amino acid sequence ofID1, and DBL2Xb, and optionally ID2a.
 10. The method according to claim2, wherein the cytotoxic moiety is selected from calicheamycin,auristatin, doxorubicin, maytansinoid, taxol, ecteinascidin,geldanamycin, methotrexate and their derivatives, cytotoxic proteinssuch as Pseudomonas exotoxin A, diphtheria toxin, ricin toxin, pokeweedantiviral protein, saporin, gelonin and functional variants, fragments,and combinations thereof.
 11. The method according to claim 2, whereinthe therapeutic or diagnostic effector moiety is an anti-inflammatoryagent.
 12. The method according to claim 2, wherein the therapeutic ordiagnostic effector moiety is CSPG4, CD44, or other proteoglycansexemplified but not limited to those in Table 1.